Low molecular weight peptidomimetic growth hormone secretagogues

ABSTRACT

The present invention comprises growth hormone releasing peptides/peptidomimetics (GHRP) capable of causing release of growth hormone from the pituitary. Compositions containing the GHRP&#39;s of this invention are used to promote growth in mammals either alone or in combination with other growth promoting compounds, especially IGF-1. In a method of this invention GHRP&#39;s in combination with IGF-1 are used to treat Type II diabetes. An exemplary compound of this invention is provided below. ##STR1##

This is a divisional of application Ser. No. 08/340,767 filed on Nov.16, 1994, now U.S. Pat. No. 5,798,337.

FIELD OF THE INVENTION

The invention relates to synthetic peptidomimetics having growth hormonereleasing activity in mammals. The peptidomimetics of this invention areused to stimulate the release of endogenous growth hormone (GH) inmammals needing elevation of serum growth hormone levels.

BACKGROUND OF THE INVENTION

GH secretion is known to be inhibited by the hypothalamic hormonesomatostatin (SS) and stimulated by GH-releasing hormone (GHRH) in allmammalian species studied including humans. In man, GH is released fromthe anterior pituitary somatotrophs in pulsatile secretory burstsoccurring about 4-8 times in each 24 hour period (Devesa, J., et al.,Trends Endocrinol Metab. 3:175-183 [1992] and Mason, W. T., et al., ActaPaediatr Suppl 388:84-92 [1993]). This episodic release pattern seems tobe optimal for inducing the physiological effects of GH since manytarget tissues appear to be more sensitive to the frequency than thetotal amount of GH arriving at the target tissue (Robinson and ClarkGrowth Hormone: Basic and Clinical Aspects Isaksson, Binder, Hall andHokfelt eds., Amsterdam, p109-127 [1987]). It is believed the episodicsecretion of GH is caused by the rhythmic alternate release of theexcitatory 44-amino acid peptide GHRH and the inhibitorytetradecapeptide SS, regulated through the "pituitary-hypothalamus axis"(see FIG. 1). Secreted GH, in turn, both directly and indirectly throughIGF-1 appears to maintain this rhythm by stimulating SS and inhibitingGHRH release. Other neurotransmitters also modulate GH release usuallyby stimulating or inhibiting SS release. Additionally, other factorsincluding exercise, sleep, glucocorticoids, thyroid hormones (e.g. TSH),sex steroids (e.g. testosterone and 17-β-estradiol), free fatty acids,amino acids (e.g. arginine and ornithine), and glucose levels furthermodulate GH release.

In addition to the two primary endogenous regulators of GH release, SSand GHRH, a number of other peptidyl/nonpeptidyl compounds have beenshown to stimulate GH release primarily through thepituitary-hypothalamus axis. These include the peptides galanin,pituitary adenylate cyclase-activation peptide (PACAP), deltasleep-inducing peptide (DSIP), and angiotensin II. These peptides,however, generally lack specificity for GH release. A number ofstructurally diverse nonpeptidyl GH secretagogues (e.g. Talipexole andClonidine) are reported to stimulate GH release in vitro and in vivo,but these compounds are believed to mediate their effect throughcholinergic, adrenergic, dopaminergic or serotonergic pathways and thusalso lack GH releasing specificity.

Apart from GHRH, the GH secretagogues having the greatest GH releasingspecificity and thus having the greatest therapeutic potential are thegrowth hormone releasing peptides/peptidomimetics (GHRP's) (Bowers, J.Pediatr. Endocrinol. 6:21-31 [1993]; and Schoen et al., Annual Reportsin Medicinal Chemistry, 28:177-186 [1993]). These compounds can activatethe pituitary-hypothalamus axis (Dickson et al., Neuroscience 53:303-306[1993]) and act directly on the pituitary somatotroph (see FIG. 1) by anindependent (non-GHRH, non-opiate and non-SS) secretory pathway.Compounds of this class can therefore be characterized by theirindependent GH releasing pathway. For example, somatotroph cellsmaximally stimulated with GHRP's can release additional GH when treatedwith GHRH and vice versa. Similarly the inhibitory effects of specificantagonists to GHRH or GHRP's have no effect on stimulation of GHrelease by the opposite secretagogue in vitro. These compounds alsoexhibit dose-dependent desensitization or attenuation of GH releaseafter continuous exposure with the same or different compounds of theGHRP class. Furthermore, structurally related biologically inactivecognate GHRP compounds capable of inhibiting GH release of a particularGHRP have no effect on GHRH agonism. These effects support theindependent pathway model and serve as experimental criteria forcompounds belonging to the GHRP class. Surprisingly, while the GHRHreceptor has been cloned in a number of species including man (Gaylin etal., Mol. Endo. 7:77-84 [1993], the GHRP receptor has remained elusive.

The paradigm compounds of the GHRP class are the syntheticmethionine-enkaphalin derived GHRP's identified by Bowers et al.,Endocrinology 106:663-667 (1980) and Momany et al., Endocrinology108:31-39 (1981). The most widely studied GHRP is referred to as"GHRP-6" (Momany et al., Endocrinology 114:1531-1536 [1984]; and Bowerset al., Endocrinology 114:1537-1545 [1984]) which has been shown; to bespecific for GH release, has no reported long term toxicity, is welltolerated, and can elevate serum GH in a dose-dependent manner in normalhumans (Bowers, J. Pediatr. Endocrinol. 6:21-31 [1993]). GHRP-6 isactive in a dose-dependent manner when administered either iv,intranasally or orally, though it is poorly absorbed orally (˜0.3%).More potent second generation hepta- and hexapeptides, "GHRP-1" and"GHRP-2" (also known as KP 102), of this class have been described morerecently, though these compounds are also expected to be poorly absorbedorally.

His-D Trp-Ala-Trp-D Phe-Lys-NH₂

(HwAWfK-NH2)

GHRP-6

Ala-His-D βNal-Ala-Trp-D Phe-Lys-NH₂

(AHbAWfK-NH2)

GHRP-1

D Ala-D βNal-Ala-Trp-D Phe-Lys-NH₂

(abAWfK-NH2)

GHRP-2

More recently, nonpeptidyl benzolactam GH secretagogues that appear touse the same alternative signal transduction pathway as GHRP-6 have beendescribed (Smith, R. G. et al., Science 260:1640-1643 [1993] and U.S.Pat. No. 5,206,235). The benzolactam L-692,429 in combination withGHRP-6 at concentrations that maximally stimulated GH release producedno additional GH release. Conversely, GHRH and L-692,429 were reportedto give a synergistic increase in GH secretion. GHRH and L-692,429 werealso reported to effect a common transient desensitation patternindicating these compounds operate through a common receptor pathway.L-692,429 is reported to be about 6-fold less potent that GHRP-6 and tobe specific for GH release, except for some in vivo ACTH and cortisolrelease. ##STR2##

A more potent analogue of L-692,429 having a potency in the ratpituitary cell assay slightly greater than GHRP-6 has also been reported(Schoen W. R. et al., Bioorg. & Medicinal Chem. Lett. 4:1117-1122[1994]). This compound, L-692,585, presumably causes GH release by thesame alternative pathway as GHRP-6. ##STR3##

A number of these compounds (e.g., "GHRP-6" and L-692,429) are reportedto be safe and effective in promoting endogenous GH release in humans,however, there remain problems with oral availability and specificity.

OBJECTS OF THE INVENTION

It is an object of this invention to provide novel GH secretagogues thatpromote the release of endogenous growth hormone in mammals. It is afurther object to provide GH secretagogues that provide a synergisticincrease in GH secretion when combined with GHRH. It is still a furtherobject of this invention to provide more potent GH secretagogues thanthose of the prior art, especially "GHRP-6", "GHRP-1", "GHRP-2",L-692,429 and L-692,585. It is a further object to provide GHsecretagogues that are specific for GH release and do not causesignificant release of other hormones, especially; LH, FSH, TSH, ACTH,prolactin, vasopressin, oxytocin, insulin and cortisol. These and otherobjects of the invention will be apparent from the followingspecification.

SUMMARY OF THE INVENTION

The objects of this invention have been achieved by providing a compoundrepresented by structural formula (I): ##STR4##

where the symbols in formula (I) define the following groups:

A is selected from ##STR5##

B is selected from ##STR6## and C₁ -C₆ alkyl substituted with ##STR7##

B may optionally be selected from the group a covalent bond, and C₁ -C₃alkyl, when L² is --N(R^(C))--Q;

C is selected from the group hydrogen, ##STR8## D--Y, and ##STR9##

D is selected from the group ##STR10## and C₁ -C₆ alkyl substituted with##STR11##

E is selected from the group ##STR12## and C₁ -C₆ alkyl substituted with##STR13##

Ar¹ and Ar² are each independently selected from substituted orunsubstituted aryl and substituted or unsubstituted heterocycle,preferably indoyl substituted with ##STR14##

Ar¹ and Ar² are independently selected from hydrogen, and C₁ -C₆ alkyl;when R^(B) or R^(C) are L¹ --Ar¹ or L² --Ar² ;

Ar³ is selected from the group ##STR15##

Ar³ is selected from hydrogen, and C₁ -C₆ alkyl; when R^(D) is L³ --Ar³,

Ar¹ together with a, Ar² together with b and Ar³ together with c, eachpair together with the carbon to which they are attached mayindependently form a 5 or 6 member carbocyclic ring;

a, b and c are independently selected from hydrogen, and C₁ -C₆ alkyl;

n and o are independently 1, 2 or 3;

L¹ is selected from --CH₂ --O--, --CH₂ --CH₂ --O--, --CH₂ --, --CH₂--CH₂ --, and --CH₂ --CH₂ --CH₂ --;

L² and L³ are independently selected from a covalent bond, --O--,--O--CH₂ --, --N(R^(C))--Q, and L¹ ;

Q is selected from the group --L² --, --S(═O)₂ --L² --, --C(═O)--,--C(═O)--O--, --CH(X)--, and --CH(X)--CH₂ --;

R^(A) is selected from the group C₀ -C₃ alkyl-heterocycle where theheterocycle comprises a mono-, bi-, or tricycle containing 5-12 ringatoms, one or two of which are heteroatoms selected from O, S, and N,provided at least one heteroatom is N, where any N atom is optionallysubstituted with R¹, C₀ -C₆ alkyl substituted with one or twosubstituents selected from the group NR² R³, imidazolinyl, pyridinyl,dihydropyridinyl, and piperidinyl;

R^(B), R^(C) and R^(D) are selected from the group R^(A), L¹ --Ar¹, L²--Ar², hydrogen, C₁ -C₆ alkyl, and halo (F, Cl, Br, I)C₁ -C₆ alkyl;

R^(A) and R^(B) together with the N to which they are bonded may form a5- or 6-member heterocycle, optionally containing one additionalheteroatom selected from O, S, and N where any N is optionallysubstituted with R¹, any carbon is optionally substituted with R⁶ andwhere the heterocycle is optionally fused to a phenyl ring, optionallysubstituted with R⁴ ;

R¹ is selected from hydrogen, C₁ -C₆ alkyl, C(═O)--C₁ -C₆ alkyl,C(═O)--NR² R³, C(═NR²)--NR² R³, C(═O)O--C₁ -C₆ alkyl, and halo(F, Cl,Br, I)C₁ -C₆ alkyl, hydroxyC₁ -C₆ alkyl, dihydroxyC₁ -C₆ alkyl;

R² and R³ are independently selected from R¹ and piperidinyl;

R² and R³ together with the N to which they are bonded may form a 5- or6-membered heterocycle, optionally containing one additional hetero atomselected from O, S, and N where any N is optionally substituted with R¹,any carbon is optionally substituted with R⁶ and where the heterocycleis optionally fused to a phenyl ring, optionally substituted with R⁴ ;

R⁴ and R⁵ are independently selected from the group hydrogen, halo(F,Cl, Br, and I), cyano, amino, amido, nitro, hydroxy, C₁ -C₆ alkyloptionally substituted with 1-3 R⁶, C₂ -C₆ alkynyl optionallysubstituted with 1-3 R⁶, C₁ -C₆ alkyloxy optionally substituted with 1-3R⁶, C₁ -C₆ acylamino optionally substituted with 1-3 R⁶, C₁ -C₆alkylcarbonyl optionally substituted with 1-3 R⁶, C₁ -C₆alkyloxycarbonyl optionally substituted with 1-3 R⁶, N--(C₁ -C₆ alkyl),N--(C₁ -C₆ acyl)amino optionally substituted with 1-3 R⁶, N--(C₁ -C₆alkyl)carboxamido optionally substituted with 1-3 R⁶, N,N-di(C₀ -C₆alkyl)amino optionally substituted with 1-3 R⁶, N,N-di(C₁ -C₆alkyl)carboxamido optionally substituted with 1-3 R⁶, C₁ -C₄perfluoroalkyl, and C₁ -C₃ perfluoroalkoxy;

R⁶ is selected from the group COOR², O(C═O)R², CONR² R³, cyano, NR² R³,NR² COR³, azido, nitro, and hydroxy;

R⁷ is selected from the group R⁶, C₆ -C₁₀ aryl optionally substitutedwith halo(F, Cl, Br, and I), cyano, amino, amido, nitro, hydroxy, C₁ -C₄perfluoroalkyl, and C₁ -C₃ perfluoroalkoxy;

X is selected from the group hydrogen, C₀ -C₆ alkyl optionallysubstituted with 1-3 R⁶, C₀ -C₆ alkyl-O--C₁ -C₆ alkyl optionallysubstituted with 1-2 R⁶, and C₁ -C₆ acyl optionally substituted with agroup selected from L² --Ar², R^(A), and R⁶ ;

Y is selected from the group --(C═O)--R^(A), C₁ -C₆ alkyl substitutedwith 1-2 R⁷, C₂ -C₆ alkynyl optionally substituted with 1-2 R⁷, C₂ -C₆alkyenyl optionally substituted with 1-2 R⁷, and C₁ -C₆ alkyloxyoptionally substituted with 1-2 R⁷,

Y and R^(D) together with the N to which they are bonded may form a 5-or 6-member heterocycle, optionally containing one additional heteroatom selected from O, S, and N where any N is optionally substitutedwith R¹, any carbon is optionally substituted with R⁷ and where theheterocycle is optionally fused to a phenyl ring;

Z is selected from the group C₁ -C₆ alkyl substituted with 1-2 R⁷, C₂-C₆ alkynyl optionally substituted with 1-2 R⁷, C₂ -C₆ alkyenyloptionally substituted with 1-2 R⁷, C₁ -C₆ alkyloxy optionallysubstituted with 1-2 R⁷ and piperidinyl; and pharmaceutically acceptablesalts thereof.

In one embodiment of the invention the compound preferably has amolecular weight between 400-650 da and is represented by formula II##STR16## where the symbols in formula II are defined as follows:

Ar¹ and Ar² are each independently selected from indoyl, ##STR17##

n and o are independently 1, 2 or 3;

L¹ is selected from --CH₂ --O--, --CH₂ --CH₂ --O--, --CH₂ --, --CH₂--CH₂ --, and --CH₂ --CH₂ --CH₂ --;

L² is selected from a covalent bond, --O--, and L¹ ;

R^(A) is selected from the group C₀ -C₃ alkyl-heterocycle, --O--C₀ -C₃alkyl-heterocycle, and --NR² --C₂ -C₆ alkyl-heterocycle, where theheterocycle comprises a mono-, bi-, or tricycle containing 5-12 ringatoms, one or two of which are heteroatoms selected from O, S, and N,provided at least one heteroatom is N, where any N atom is optionallysubstituted with R¹, C₀ -C₆ alkyl substituted with one or twosubstituents, O--C₂ -C₆ alkyl substituted with one or two substituents,and NR² --C₂ -C₆ alkyl substituted with one or two substituents wherethe substituents are selected from the group NR² R³, imidazolinyl,pyridinyl, dihydropyridinyl, and piperidinyl;

R^(B) and R^(C) are selected from the group hydrogen, C₁ -C₆ alkyloptionally substituted with a group selected from NR² R³, and phenyl-C₁-C₃ -alkyl-NR² R³, and halo(F, Cl, Br, I)C₁ -C₆ alkyl;

R¹ is selected from hydrogen, C₁ -C₆ alkyl, C(═O)--C₁ -C₆ alkyl,C(═O)--NR² R³, C(═NR²)--NR² R³, C(═O)O--C₁ -C₆ alkyl, and halo(F, Cl,Br, I)C₁ -C₆ alkyl, C₁ -C₆ alkoxyalkyl or (hydroxylalkyl);

R² and R³ are independently selected from hydrogen, C₁ -C₆ alkyl,piperidinyl, and halo(F, Cl, Br, I)C₁ -C₆ alkyl;

R² and R³ together with the N to which they are bonded may form a 5- or6-membered heterocycle, optionally containing one additional hetero atomselected from O, S, and N where any N is optionally substituted with R¹,any carbon is optionally substituted with R⁶ and where the heterocycleis optionally fused to a phenyl ring, optionally substituted with R⁴ ;

R⁴ and R⁵ are independently selected from the group hydrogen, halo(F,Cl, Br, and I), cyano, amino, amido, nitro, hydroxy, C₁ -C₆ alkyloptionally substituted with 1-3 R⁶, C₂ -C₆ alkynyl optionallysubstituted with 1-3 R⁶, C₁ -C₆ alkyloxy optionally substituted with 1-3R⁶, C₁ -C₆ acylamino optionally substituted with 1-3 R⁶, C₁ -C₆alkylcarbonyl optionally substituted with 1-3 R⁶, C₁ -C₆alkyloxycarbonyl optionally substituted with 1-3 R⁶, N--(C₁ -C₆ alkyl),N--(C₁ -C₆ acyl)amino optionally substituted with 1-3 R⁶, N--(C₁ -C₆alkyl)carboxamido optionally substituted with 1-3 R⁶, N,N-di(C₀ -C₆alkyl)amino optionally substituted with 1-3 R⁶, N,N-di(C₁ -C₆alkyl)carboxamido optionally substituted with 1-3 R⁶, C₁ -C₄perfluoroalkyl, and C₁ -C₃ perfluoroalkoxy;

R⁶ is selected from the group COOR², O(C═O)R², CONR² R³, cyano, NR² R³,NR² COR³, azido, nitro, and hydroxy;

X is selected from the group hydrogen, oxo (═O), COOR², CONR² R³, C₀ -C₆alkyl-O--C₁ -C₆ alkyl optionally substituted with 1-2 R⁶, and C₁ -C₆alkyl optionally substituted with 1-2 R⁶ ; and pharmaceuticallyacceptable salts thereof.

Alternative compounds of this embodiment may be represented by formulaIIa-IIg ##STR18##

where Ar¹, Ar², R^(B), R^(C), R¹, R², R³, R⁶, Q and X are defined above,and p is 0, 1 or 2.

Optionally the Ar¹, Ar², R^(B), R^(C), R¹, R², R³, R⁶ and X are definedas follows:

Ar¹ and Ar² are each independently selected from indoyl, and ##STR19##

R^(B) and R^(C) are selected from the group hydrogen, and methyl;

R¹ is selected from hydrogen, C₁ -C₆ alkyl, C₂ -C₆ alkyl substitutedwith 1 or 2 hydroxy groups, C(═O)--C₁ -C₆ alkyl, C(═O)--NR² R³,C(═NR²)--NR² R³, C(═O)O--C₁ -C₆ alkyl, and halo (F, Cl, Br, I)C₁ -C₆alkyl;

R² and R³ are independently selected from hydrogen, C₁ -C₆ alkyl,piperidinyl, and halo(F, Cl, Br, I)C₁ -C₆ alkyl;

R² and R³ together with the nitrogen to which they are attached may formpiperidinyl, pyrroylidinyl, piperazinyl, and morpholinyl;

R⁶ is selected from the group COOR², O(C═O)R², CONR² R³, cyano, NR² R³,NR² COR³, azido, nitro, and hydroxy;

X is selected from the group hydrogen, oxo (═O), COOR², CONR² R³, C₀ -C₆alkyl-O--C₁ -C₆ alkyl optionally substituted with 1-2 R⁶, and C₁ -C₆alkyl optionally substituted with 1-2 R⁶ ; and pharmaceuticallyacceptable salts thereof.

In an alternative embodiment of the invention the compound isrepresented by structural formula III-IIIi ##STR20##

where the symbols in formula III-IIIi are defined as follows:

Ar¹ and Ar² are each independently selected from indoyl, ##STR21##

n and o are independently 1, 2 or 3;

L¹ and L² are independently selected from --CH₂ --O--, --CH₂ --CH₂ --CH₂--O--CH₂, --CH₂ --CH₂ --, and --CH₂ --CH₂ --CH₂ --;

R^(A) is selected from the group C₀ -C₃ alkyl-heterocycle, --O--C₀ -C₃alkyl-heterocycle, and --NR² --C₂ -C₆ alkyl-heterocycle, where theheterocycle comprises a mono-, bi-, or tricycle containing 5-12 ringatoms, one or two of which are heteroatoms selected from O, S, and N,provided at least one heteroatom is N, where any N atom is optionallysubstituted with R¹, C₀ -C₆ alkyl substituted with one or twosubstituents, O--C₂ -C₆ alkyl substituted with one or two substituents,and NR² --C₂ -C₆ alkyl substituted with one or two substituents wherethe substituents are selected from the group NR² R³, imidazolinyl,pyridinyl, dihydropyridinyl, and piperidinyl;

R^(B), R^(C) and R^(D) are selected from the group hydrogen, C₁ -C₆alkyl optionally substituted with a group selected from NR² R³, andphenyl-C₁ -C₃ -alkyl-NR² R³, and halo(F, Cl, Br, I)C₁ -C₆ alkyl;

R¹ is selected from hydrogen, C₁ -C₆ alkyl, C(═O)--C₁ -C₆ alkyl,hydroxyalkyl C(═O)--NR² R³, C(═NR²)--NR² R³, C(═O)O--C₁ -C₆ alkyl, andhalo(F, Cl, Br, I)C₁ -C₆ alkyl;

R² and R³ are independently selected from hydrogen, C₁ -C₆ alkyl,piperidinyl, and halo(F, Cl, Br, I)C₁ -C₆ alkyl;

R² and R³ together with the N to which they are bonded may form a 5- or6-membered heterocycle, optionally containing one additional hetero atomselected from O, S, and N where any N is optionally substituted with R¹,any carbon is optionally substituted with R⁶ and where the heterocycleis optionally fused to a phenyl ring, optionally substituted with R⁴ ;

R⁴ and R⁵ are independently selected from the group hydrogen, halo(F,Cl, Br, and I), cyano, amino, amido, nitro, hydroxy, C₁ -C₆ alkyloptionally substituted with 1-3 R⁶, C₂ -C₆ alkynyl optionallysubstituted with 1-3 R⁶, C₁ -C₆ alkyloxy optionally substituted with 1-3R⁶, C₁ -C₆ acylamino optionally substituted with 1-3 R⁶, C₁ -C₆alkylcarbonyl optionally substituted with 1-3 R⁶, C₁ -C₆alkyloxycarbonyl optionally substituted with 1-3 R⁶, N--(C₁ -C₆ alkyl),N--(C₁ -C₆ acyl)amino optionally substituted with 1-3 R⁶, N--(C₁ -C₆alkyl)carboxamido optionally substituted with 1-3 R⁶, N,N-di(C₀ -C₆alkyl)amino optionally substituted with 1-3 R⁶, N,N-di(C₁ -C₆alkyl)carboxamido optionally substituted with 1-3 R⁶, C₁ -C₄perfluoroalkyl, and C₁ -C₃ perfluoroalkoxy;

R⁶ is selected from the group COOR², O(C═O)R², CONR² R³, cyano, NR² R³,NR² COR³, azido, nitro, and hydroxy;

R⁷ is selected from the group R⁶, and C₆ -C₁₀ aryl optionallysubstituted with halo(F, Cl, Br, and I), cyano, amino, amido, nitro,hydroxy, C₁ -C₄ perfluoroalkyl, and C₁ -C₃ perfluoroalkoxy;

Y is selected from the group C₁ -C₆ alkyl substituted with 1-2 R⁷, C₂-C₆ alkynyl optionally substituted with 1-2 R⁷, C₂ -C₆ alkyenyloptionally substituted with 1-2 R⁷, and C₁ -C₆ alkyloxy optionallysubstituted with 1-2 R⁷, and pharmaceutically acceptable salts thereof.

In a further alternative embodiment of this invention the compound isrepresented by structural formula IV ##STR22##

where the symbols of formula IV are defined as follows:

Ar¹ and Ar² are each independently selected from indoyl, ##STR23##

Ar³ is selected from the group ##STR24##

n and o are independently 1, 2 or 3;

R^(A) is selected from the group C₀ -C₃ alkyl-heterocycle, --O--C₀ -C₃alkyl-heterocycle, and --NR² --C₂ -C₆ alkyl-heterocycle, where theheterocycle comprises a mono-, bi-, or tricycle containing 5-12 ringatoms, one or two of which are heteroatoms selected from O, S, and N,provided at least one heteroatom is N, where any N atom is optionallysubstituted with R¹, C₀ -C₆ alkyl substituted with one or twosubstituents, O--C₂ -C₆ alkyl substituted with one or two substituents,and NR² --C₂ -C₆ alkyl substituted with one or two substituents wherethe substituents are selected from the group NR² R³, imidazolinyl,pyridinyl, dihydropyridinyl, and piperidinyl;

R^(B), R^(C), R^(D), and R^(E) are selected from the group hydrogen, C₁-C₆ alkyl optionally substituted with a group selected from NR² R³, andphenyl-C₁ -C₃ -alkyl-NR² R³, and halo(F, Cl, Br, I)C₁ -C₆ alkyl;

R¹ is selected from hydrogen, C₁ -C₆ alkyl, C(═O)--C₁ -C₆ alkyl,C(═O)-hydroxyalkyl NR² R³, C(═NR²)--NR² R³, C(═O)O--C₁ -C₆ alkyl, andhalo(F, Cl, Br, I)C₁ -C₆ alkyl;

R² and R³ are independently selected from hydrogen, C₁ -C₆ alkyl,piperidinyl, and halo(F, Cl, Br, I)C₁ -C₆ alkyl;

R² and R³ together with the N to which they are bonded may form a 5- or6-membered heterocycle, optionally containing one additional hetero atomselected from O, S, and N where any N is optionally substituted with R¹,any carbon is optionally substituted with R⁶ and where the heterocycleis optionally fused to a phenyl ring, optionally substituted with R⁴ ;

R⁴ and R⁵ are independently selected from the group hydrogen, halo(F,Cl, Br, and I), cyano, amino, amido, nitro, hydroxy, C₁ -C₆ alkyloptionally substituted with 1-3 R⁶, C₂ -C₆ alkynyl optionallysubstituted with 1-3 R⁶, C₁ -C₆ alkyloxy optionally substituted with 1-3R⁶, C₁ -C₆ acylamino optionally substituted with 1-3 R⁶, C₁ -C₆alkylcarbonyl optionally substituted with 1-3 R⁶, C₁ -C₆alkyloxycarbonyl optionally substituted with 1-3 R⁶, N--(C₁ -C₆ alkyl),N--(C₁ -C₆ acyl)amino optionally substituted with 1-3 R⁶, N--(C₁ -C₆alkyl)carboxamido optionally substituted with 1-3 R⁶, N,N-di(C₀ -C₆alkyl)amino optionally substituted with 1-3 R⁶, N,N-di(C₁ -C₆alkyl)carboxamido optionally substituted with 1-3 R⁶, C₁ -C₄perfluoroalkyl, and C₁ -C₃ perfluoroalkoxy;

R⁶ is selected from the group COOR², O(C═O)R², CONR² R³, cyano, NR² R³,NR² COR³, azido, nitro, and hydroxy;

R⁷ is selected from the group R⁶, C₆ -C₁₀ aryl optionally substitutedwith halo(F, Cl, Br, and I), cyano, amino, amido, nitro, hydroxy, C₁ -C₄perfluoroalkyl, and C₁ -C₃ perfluoroalkoxy;

Z is selected from the group C₁ -C₆ alkyl substituted with 1-2 R⁷, C₂-c₆ alkynyl optionally substituted with 1-2 R⁷, C₂ -C₆ alkyenyloptionally substituted with 1-2 R⁷, and C₁ -C₆ alkyloxy optionallysubstituted with 1-2 R⁷,

Z and R^(E) together with the N to which they are bonded may form a 5-or 6-membered heterocycle, optionally containing one additional heteroatom selected from O, S, and N where any N is optionally substitutedwith R¹, any carbon is optionally substituted with R⁷ and where theheterocycle is optionally fused to a phenyl ring; and pharmaceuticallyacceptable salts thereof.

An optional compound of this embodiment is represented by structuralFormula (IVa) ##STR25##

where the symbols in formula IVa are defined as follows:

R^(B), R^(C), R^(D) and R^(E) are selected from the group hydrogen, andC₁ -C₆ alkyl;

Ar¹ and Ar² are each independently selected from indoyl, and ##STR26##

Ar³ is selected from the group ##STR27##

R^(F) is selected from the group OH, C₁ -C₄ alkyloxy, NR⁵ R⁶, and 1 to 4α-amino acid residues;

R⁴ is selected from hydrogen, halo(F, Cl, Br, and I), cyano, amino,amido, nitro, hydroxy, C₁ -C₄ alkoxy, C₁ -C₄ perfluoroalkyl, and C₁ -C₃perfluoroalkoxy;

R⁵ and R⁶ are independently selected from hydrogen, and C₁ -C₆ alkyl;and pharmaceutically acceptable salts thereof.

In still another embodiment of this invention the compound is referredto as a "retroinverso" of the compound of formula II and is representedby formula V ##STR28##

where Ar¹, Ar², L¹, L², R^(A), R^(B), R^(C) and X are defined above forthe compound of formula I.

The invention further comprises a pharmaceutical composition comprisinga pharmaceutically acceptable excipient and any of the compoundsrepresented by structural formula I-V. Additionally the inventionprovides a method for increasing the level of endogenous growth hormonein a mammal comprising administering to the mammal a pharmaceuticallyeffective amount of the forgoing composition to the mammal. The methodfurther comprises administering the composition in combination with agrowth factor selected from; growth hormone releasing hormone (GHRH),insulin like growth factor-1 (IGF-1), and insulin like growth factor-2(IGF-2). In an alternative method of this invention GHRP's representedby formulae I-V, as well as other GHRP's, are used in combination withIGF-1 to treat diseases in which long term IGF-1 is indicated includingbut not limited to Type II diabetes.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. A cartoon showing regulation of growth hormone (GH) release bythe "pituitary-hypothalamus axis" and the alternative "GHRP pathway".Some of the stimulatory (+) and inhibitory (-) effects that growthhormone releasing hormone/factor (GHRH/F), growth hormone releasingpeptides/peptidomimetics (GHRP's ), somatostatin (SS), GH, andinsulin-like growth factor 1 (IGF-1) have on selected glands, organs,and tissues are also shown.

FIG. 2. A representative rat "pit" cell assay dose response of GHrelease over a 15 min. exposure to increasing concentrations of(inip)-bbFK-NH₂. GH release is significantly (P<0.05) increased at 0.3nM and reaches a plateau by 1 nM with an EC₅₀ of 0.16 nM (see rightpanel for data points and curve used to calculate EC₅₀). The mean (n=3)EC₅₀ for inip-bbFK-NH₂ was 0.18±0.04 nM, over 30-fold more potent thanHwAWfK-NH₂ ("GHRP-6") (6.2±1.5 nM; n=5).

FIG. 3. Demonstration that (inip)-bbFK-NH₂ acts at the proposed "GHRPreceptor". Rat "pit" cell challenges were carried out using combinationsof GHRP-6, (inip)-bbFK-NH₂ and GHRH. GHRP-6 and (inip)-bbFK-NH₂ (100 nM)both caused 3-fold increases in GH levels over control (ctrl), but noadditional or synergistic increase was observed when used in combination(solid black bars). In contrast, both GHRP-6 and (inip)-bbFK-NH₂ showsynergy with 10 nM GHRH indicating that neither GHRP-6 nor(inip)-bbFK-NH₂ act via the GHRH receptor (cross hatched bars).

FIG. 4. Desensitization effect of the "GHRP receptor" upon challengingrat pituitary cells with three sequential 15 min. incubations with fresh(inip)-bbFK-NH₂. The GH release was decreased after the second 15 min.incubation (total 30 min. exposure to (inip)-bbFK-NH₂) and nosignificant GH release compared to control occurred during the finalchallenge with (inip)-bbFK-NH₂. However when GHRH (10 nM) was added tothe next 15 min. incubation, a significant GH response occurred,consistent with the separate receptor model.

FIG. 5. Somatostatin suppression of GHRP-stimulated GH release.(Inip)-bbFK-NH₂ at 0.1, 1, 10 and 100 nM significantly elevates GHrelease (solid black bars). Coincubation of (inip)-bbFK-NH₂ with 20 nMsomatostatin suppressed this release (crosshatched bars).

FIG. 6. Antagonism of (inip)-bbFK-NH₂ induced GH release in the absence(solid black bars) and presence of 10 μM of the "GHRP-6" antagonistHwkWfK-NH₂ (crosshatched bars).

FIG. 7. Specificity for GH release compared to prolactin release inducedwith (inip)-bbFK-NH₂. GH and prolactin levels in the same media fromcells challenged with 100 nM (inip)-bbFK-NH₂. GH release was 3-foldwhile prolactin levels increased 1.6 fold.

FIG. 8. TSH, FSH and LH release induced with (inip)-bbFK-NH₂. Pituitarycells challenged with (inip)-bbFK-NH₂ had no effect on these hormoneconcentrations.

FIG. 9. ACTH release induced with corticotrophin releasing factor (CRF)or (inip)-bbFK-NH₂. ACTH levels rose 3-fold when stimulated with CRF butno significant change was observed with 100 nM (inip)-bbFK-NH₂.

FIGS. 10A-10D. Ca⁺⁺ release in pituitary cells by (inip)-bbFK-NH₂. BasalCa⁺⁺ in Indol-1 AM loaded pituitary cells after 4-day monolayer culture(FIG. 10A and FIG. 10C). Twenty-one (21) seconds after (inip)-bbFK-NH₂addition, the increased Ca⁺⁺ flux in FIG. 10D is demonstrated by lighterareas in some of the cells of this heterologous population. Addition ofvehicle did not change Ca⁺⁺ levels (FIG. 10B).

FIG. 11. Dose dependent GH release with (inip)bb(feg). GH release by ratpituitary cells to increasing concentrations of (inip)bb(feg) (leftpanel) over a 15 minute incubation. Right panel shows data points andcurve used to calculate the EC₅₀ of 0.09 nM.

FIG. 12. Demonstration that (inip)bb(feg) acts at the proposed "GHRPreceptor". GH response to GHRP-6 (100 nM) and (inip)bb(feg) (100 nM) wassignificantly greater than control but GH release was not synergisticwhen both were added in combination. GHRH (100 nM) elicited a mild GHresponse which was synergistic in combination with either GHRP-6 or(inip)bb(feg).

FIG. 13. Somatostatin suppression of (inip)bb(feg)-stimulated GHrelease. GH release with 100 nM (inip)bb(feg) was totally suppressed inthe presence of 20 nM somatostatin.

FIG. 14. Desensitization of the "GHRP receptor" upon challenging ratpituitary cells with three sequential 15 min. incubations with fresh(inip)bb(feg). GH release from the same pituitary cells over threesequential 15 minute incubations with (inip)bb(feg) (100 nM). After atotal of 45 minutes, GH release was markedly decreased in response to(inip)bb(feg) but these cells were able to release more GH in responseto a final 15 minute incubation with GHRH (10 nM).

FIG. 15. Dose dependent GH release with (inip)b(wol). GH release by ratpituitary cells to increasing concentrations of (inip)b(wol) (leftpanel) over a 15 minute incubation. Right panel shows the data pointsand curve used to calculate the EC₅₀ of 3.9 nM.

FIG. 16. Demonstration that (inip)b(wol) acts at the proposed "GHRPreceptor". GH response to GHRP-6 (100 nM) and (inip)b(wol) (100 nM) wassignificantly greater than control but GH release was not synergisticwhen both were added in combination. GHRH (100 nM) elicited a mild GHresponse which was synergistic in combination with either GHRP-6 or(inip)b(wol).

FIG. 17. Somatostatin suppression of (inip)b(wol)-stimulated GH release.GH release to 100 nM (inip)b(wol) was totally suppressed in the presenceof 20 nM somatostatin.

FIG. 18. Desensitization effect of the "GHRP receptor" upon challengingrat pituitary cells with three sequential 15 min. incubations with fresh(inip)b(wol). GH release from the same pituitary cells over threesequential 15 minute incubations with (inip)b(wol) (100 nM). After atotal of 45 minutes, no significant release of GH was observed inresponse to (inip)b(wol) but these cells were able to release GH inresponse to a final 15 minute incubation with GHRH (10 nM).

FIG. 19. Body weight gain in normal adult female rats in response toexcipient (open squares), three doses of (inip)bbFK-NH2 (4 μg/d opencircles, 20 μg/d open triangles, 100 μg/d half-filled diamonds), or onedose of GHRH (600 μg/d, filled squares); delivery was by subcutaneousminipump infusion for 14 days. There was a dose-related weight gain inresponse to (inip)bbFK-NH2, that at 20 μg/d reached the response toGHRH. Means and standard errors are shown.

FIG. 20. Body weight gain in normal adult female rats treated for 14days with excipient (open bar), two doses of (inip)bbFK-NH2 givensubcutaneously by injection (shaded bar, 100 μg/d; solid bar, 20 μg/d)or infusion (lightly hatched bar, 100 μg/d; heavily hatched bar, 20μg/d). Injections of (inip)bbFK-NH2 were more effective than infusions.Means and standard errors are shown.

FIG. 21. Body weight gain in normal adult female rats treated for 14days with excipient (open squares), or (inip)bbFK-NH2 given bysubcutaneous injection (100 μg/d; open triangles) or infusion (100 μg/d;open circles). Injections of (inip)bbFK-NH2 produced a maintained growthresponse; infusions gave a large initial response that was notmaintained. Means and standard errors are shown.

FIG. 22. Body weight gain in lean (open circles) and obese Type IIZucker Diabetic Fatty (ZDF) male rats treated subcutaneously for 24 dayswith excipient (solid circles), recombinant human growth hormone (rhGH,large open squares, 500 μg/d), recombinant human insulin-like growthfactor-1 (rhIGF-1, small squares, 758 μg/d), (inip)bbFK-NH2 given byinjection (100 μg/d; open triangles), the combination of rhGH plusrhIGF-1 (solid squares), or the combination of rhIGF-1 plus(inip)bbFK-NH2 (solid triangles). The combination of (inip)bbFK-NH2 plusrhIGF-1 produced a maintained growth response equal to that of rhIGF-1plus rhGH. Means and standard errors are shown.

FIG. 23. Body weight gain in obese Type II Zucker Diabetic Fatty (ZDF)male rats treated subcutaneously for 7 days with excipient (solidcircles), recombinant human insulin-like growth factor-1 (rhIGF-1, opencircles, 758 μg/d), (inip)bbFK-NH2 given by injection (100 μg/d; opentriangles), or the combination of rhIGF-1 and (inip)bbFK-NH2 (solidtriangles). The combination of (inip)bbFK-NH2 plus rhIGF-1 produced amaintained growth response that was at least additive compared to eachagent given alone. Means and standard errors are shown.

FIG. 24. Basal blood glucose levels obtained weekly in lean (smallsquares) and obese Type II Zucker Diabetic Fatty (ZDF) male rats treatedfor 3 weeks subcutaneously with excipient (controls, solid circles),recombinant human growth hormone (rhGH, open squares, 500 μg/d),recombinant human insulin-like growth factor-1 (rhIGF-1, open circles,758 μg/d), (inip)bbFK-NH2 given by injection (100 μg/d; open triangles),or the combination of rhGH and rhIGF-1 (solid squares), or thecombination of rhIGF-1 and (inip)bbFK-NH2 (solid triangles). When givenalone, or in combination with rhIGF-1, (inip)bbFK-NH2 had lesser effecton blood glucose (diabetogenic effect) than rhGH at doses with similarsomatogenic effects (FIG. 22). Means and standard errors are shown.

FIG. 25. Blood glucose concentrations following an intravenous insulinchallenge (insulin tolerance test) in lean control male rats (open bar),obese Type II Zucker Diabetic Fatty (ZDF) rats treated subcutaneouslywith excipient (solid bar), recombinant human growth hormone (rhGH,light left-right ascending hatching, 500 μg/d), recombinant humaninsulin-like growth factor-1 (rhIGF-1, light shaded bar, 758 μg/d),(inip)bbFK-NH2 given by injection (100 μg/d; light left-right descendinghatching), or the combination of rhGH and rhIGF-1 (heavy left-rightascending hatching), or the combination of rhIGF-1 and (inip)bbFK-NH2(heavy left-right descending hatching). When given alone, or incombination with rhIGF-1, (inip)bbFK-NH2 had a greatly reduced effect oninsulin sensitivity (diabetogenic effect) than rhGH at doses withsimilar somatogenic effects (FIG. 22). Means and standard errors areshown.

FIG. 26. Daily body weight gains in normal adult female rats treated for7 days with twice daily subcutaneous injections of excipient (opensquares), growth hormone releasing hormone (open triangles), HwAWfK(solid squares), (inip)bbF-NH2 (open circles), (inip)b nmb bam (solidcircles), or L-692,585 (solid triangles). Significant weight gainoccurred after treatment with all molecules except L-692,585. Means andstandard errors are shown.

FIG. 27. Total body weight gain in normal adult rats treated for 7 dayswith twice daily subcutaneous injections of excipient (solid bar),infusions of recombinant human insulin-like growth factor-1 (rhIGF-1,broad hatching), or the combinations of rhIGF-1 plus growth hormonereleasing hormone (light shading), HwAWfK plus rhIGF-1 (narrowhatching), (inip)bbF-NH2 plus rhIGF-1(open bar), (inip)b nmb bam plusrhIGF-1 (dark shading), or L-692,585 plus rhIGF-1 (horizontal lines).Weight gains tended to be greater after combination treatment. Means andstandard errors are shown.

DETAILED DESCRIPTION OF THE INVENTION

A. Definitions

Terms used in the claims and specification are defined as set forthbelow unless otherwise specified.

The terms growth hormone releasing hormone (GHRH) or factor (GHRF/GRF)are used interchangeably and refer to the endogenous hypothalamic GHsecretagogue, from any species, having the capability of binding to thepituitary somatotroph and inducing a rapid dose-dependent release of GHand biologically active analogs thereof. Included in this definitionare; GHRH(1-44), GHRH(1-43), GHRH(1-40), and GHRH (1-29). Other examplesof GHRH analogs are described in U.S. Pat. No. 4,622,312.

       1              5                   10                   15                   H-Tyr-Ala-Aap-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Lys-Val-Leu-Gly-                 -    16             20                  25                 30                  Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asp-Ile-Met-Ser-Arg-Gln-                 -    31             35                  40              44                     Gln-Gly-Glu-Ser-Asn-Gln-Glu-Arg-Gly-Ala-Arg-Ala-Arg-Leu-NH.sub.2                                                  -              Growth Hormone                                               Releasing Hormone                          -                         (GHRH)                                       

The term somatostatin (SS) refers to the inhibitory hypothalamictetradecapeptide capable of antagonizing in a dose-dependent manner theGH-releasing effect of GHRH.

       1               5                                                            H-Ala-Gly-Cys-Lys-Asn-Phe-Phe-Trp                                                        |                   |                                      Cys-Ser-Thr-Phe-Thr-Lys                                                       14              10                                                            Somatostatin                                                                     (SS)                                                       

As used herein, "IGF-1" refers to insulin-like growth factor from anyspecies, including bovine, ovine, porcine, equine, avian, and preferablyhuman, in native-sequence or in variant form, and from any source,whether natural, synthetic, or recombinant. Preferred herein for animaluse is that form of IGF-1 from the particular species being treated,such as porcine IGF-1 to treat pigs, ovine IGF-1 to treat sheep, bovineIGF-1 to treat cattle, etc. Preferred herein for human use is humannative-sequence, mature IGF-1, more preferably without a N-terminalmethionine, prepared, for example, by the process described in EP230,869 published Aug. 5, 1987; EP 128,733 published Dec. 19, 1984; orEP 288,451 published Oct. 26, 1988. More preferably, this nativesequence IGF-1 is recombinantly produced and is available fromGenentach, Inc., South San Francisco, Calif. for clinicalinvestigations. Also preferred for use is IGF-1 that has a specificactivity greater than about 14,000 units/mg as determined byredioreceptor assay using placenta membranes, such as that availablefrom KabiGen AB, Stockholm, Sweden.

The most preferred IGF-1 variants are those described in U.S. Pat. No.5,077,276 issued Dec. 31, 1991, in PCT WO 87/01038 published Feb. 26,1987 and in PCT WO 89/05822 published Jun. 29, 1989, i.e., those whereinat least the glutamic acid residue is absent at position 3 from theN-terminus of the mature molecule or those having a deletion of up tofive amino acids at the N-terminus. The most preferred variant has thefirst three amino acids from the N-terminus deleted (variouslydesignated as brain IGF, tIGF-1, des(1-3)IGF-1, or des-IGF-1).

The term "GHRP" as used herein refers to compounds that cause release ofendogenous GH in a dose-dependent manner, where such release issynergized by GHRH but not by other GHRP's such as GHRP-6, and wheresuch release causes a desensitization after continuous exposure to theGHRP while maintaining the ability to respond to GHRH.

The term "C_(n) -C_(m) alkyl" means a cyclic or linear, branched orunbranched, saturated aliphatic hydrocarbon radical, having the numberof carbon atoms specified, where m and n are zero or integersidentifying the range of carbon atoms contained in the alkyl group. Whenn is zero (0) the term becomes a chemical bond, usually a covalent bond.Examples of alkyl radicals include methyl, ethyl, n-propyl,isopropyl(iPr), n-butyl, iso-butyl, sec-butyl, tert-butyl(tBu),n-pentyl, 2-methylbutyl, 2,2-dimethylpropyl, n-hexyl, 2-methylpentyl,2,2-dimethylbutyl, n-heptyl, 2-methylhexyl, cyclohexyl, and the like.The terms "lower alkyl" and "C₁ -C₆ alkyl" are synonymous and usedinterchangeably.

The term "C₂ -C_(m) alkenyl" means a cyclic or linear, branched orunbranched hydrocarbon radical containing at least one carbon--carbondouble bond, having the number of carbon atoms specified, each doublebond being independently cis, trans, E or Z, or a non-geometric isomer.

The term "C₂ -C_(m) alkynyl" means a cyclic or linear, branched orunbranched hydrocarbon radical containing at least one carbon--carbontriple bond, having the number of carbon atoms specified,

The terms "C₁ -C₁₂ acyloxy" or "C₁ -C₁₂ alkanoyloxy" are usedinterchangeably and denote herein groups of the formula C₀ -C₁₂alkyl-C(═O)--O-- such as; formyloxy, acetoxy, propionyloxy, butyryloxy,pentanoyloxy, hexanoyloxy, heptanoyloxy, and the like.

The term "N,N-di(C₀ -C₆)alkylamino" denotes herein groups of the formula(C₀ -C₆ alkyl)₂ --N-- where both, one or none of the hydrogen atoms ofH₂ N-- are substituted with C₁ -c₆ alkyl.

The term "N--(C₁ -C₆ alkyl), N--(C₁ -C₆ acyl)amino" denotes herein anamino group where one hydrogen is substituted with a C₁ -C₆ alkyl groupand the other hydrogen is substituted with a C₁ -C₆ acyl group.

The terms "C₁ -c₆ alkyloxycarbonyl" and "C₁ -C₆ carboalkoxy" are usedinterchangeably herein and denote groups of the formula C₁ -c₆alkyl-O--C(═O)--.

The terms "N--(C₁ -C₆ alkyl)carboxamido" and "N--(C₁ -C₆alkyl)-aminocarbonyl" are used interchangeably herein and denote groupsof the formula C₁ -C₆ alkyl-NH--C(═O)--.

The terms "C₁ -C₁₂ alkylcarbonyl", "C₁ -C₁₂ alkanoyl" and "C₁ -C₁₂ acyl"are used interchangeably herein and denote groups of the formula C₀ -C₁₂alkyl-C(═O)-- and encompass groups such as formyl, acetyl, propionyl,butyryl, pentanoyl, hexanoyl, heptanoyl, benzoyl and the like.

The term "C₁ -C₆ acylamino" denotes groups of the formula C₁ -c₆alkyl-C(═O)--NH--.

The terms "C₁ -C₁₂ alkyloxy" and "C₁ -C₁₂ substituted alkyloxy" denoteC₁ -C₁₂ alkyl and C₁ -C₁₂ substituted alkyl groups, respectively,attached to an oxygen which is in turn the point of attachment for thealkyloxy or substituted alkyloxy group to the group or substituentdesignated (e.g. C₁ -C₁₂ alkyl-O--). These include groups such asmethoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, t-butoxy,cyclohexyloxy and like groups.

The term "aryl" when used alone means a homocyclic hydrocarbon aromaticradical, whether or not fused, having the number of carbon atomsdesignated or if none are designated--from 6 to 14. Aromatic radicalsmay be mononuclear or polynuclear. Examples of aryl groups includephenyl, napthyl anthranyl, phenanthranyl, azulyl and the like.

Preferred aryl groups include phenyl, napthyl, biphenyl, phenanthrenyl,naphthacenyl, and the like (see e.g. Lang's Handbook of Chemistry (Dean,J. A., ed) 13^(th) ed. Table 7-2 [1985]).

Optionally the "aryl" is substituted with one or more substituentsusually designated by a group "--R^(n) ", where n is any integer.Examples of substituted phenyl groups include mono- or di(halo)phenylgroups such as 4-chlorophenyl, 2,6-dichlorophenyl, 2,5-dichlorophenyl,3,4-dichlorophenyl, 3-chlorophenyl, 3-bromophenyl, 4-bromophenyl,3,4-dibromophenyl, 3-chloro-4-fluorophenyl, 2-fluorophenyl and the like;mono- or di(hydroxy)phenyl groups such as 4-hydroxyphenyl,3-hydroxyphenyl, 2,4-dihydroxyphenyl, the protected-hydroxy derivativesthereof and the like; nitrophenyl groups such as 3- or 4-nitrophenyl;cyanophenyl groups, for example, 4-cyanophenyl; mono- or di(loweralkyl)phenyl groups such as 4-methylphenyl, 2,4-dimethylphenyl,2-methylphenyl, 4-(isopropyl)phenyl, 4-ethylphenyl, 3-(n-propyl)phenyland the like; mono or di(alkoxy)phenyl groups, for example,2,6-dimethoxyphenyl, 4-methoxyphenyl, 3-ethoxyphenyl,4-(isopropoxy)phenyl, 4-(t-butoxy)phenyl, 3-ethoxy-4-methoxyphenyl andthe like; 3- or 4-trifluoromethylphenyl; mono- or dicarboxyphenyl or(protected carboxy)phenyl groups such 4-carboxyphenyl; mono- ordi(hydroxymethyl)phenyl or (protected hydroxymethyl)phenyl groups suchas 3-(protected hydroxymethyl)phenyl or 3,4-di(hydroxymethyl)phenyl,2,3- and 3,4-methylene dioxy; mono- or di(aminomethyl)phenyl or(protected aminomethyl)phenyl groups such as 2-(aminomethyl)phenyl or2,4-(protected aminomethyl)phenyl; or mono- ordi(N-(methylsulfonylamino))phenyl groups such as3-(N-methylsulfonylamino))-phenyl. Also, the term "substituted phenyl"represents disubstituted phenyl groups wherein the substituents aredifferent, for example, 3-methyl-4-hydroxyphenyl,3-chloro-4-hydroxyphenyl, 2-methoxy-4-bromophenyl,4-ethyl-2-hydroxyphenyl, 3-hydroxy-4-nitrophenyl,2-hydroxy-4-chlorophenyl and the like. Preferred substituted phenylgroups include the 2- and 3-trifluoromethylphenyl, 4-fluoro orchlorophenyl the 4-hydroxyphenyl, the 2-aminomethylphenyl and the3-(N-(methylsulfonylamino))phenyl groups.

The term "arylalkyl" means one, two, or three aryl groups having thenumber of carbon atoms designated, appended to an alkyl radical havingthe number of carbon atoms designated including but not limited to;benzyl, napthylmethyl, phenethyl, benzyhydryl (diphenylmethyl), trityl,and the like. A preferred arylalkyl group is the benzyl group.

The term "substituted C₆ -C₁₂ aryl-C₁ -C₆ alkyl" denotes a C₁ -C₆ alkylgroup substituted at any carbon with a C₆ -C₁₂ aryl group bonded to thealkyl group through any aryl ring position and substituted on the C₁ -C₆alkyl portion with one, two or three groups chosen from halogen(F, Cl,Br, I), hydroxy, protected hydroxy, amino, protected amino, C₁ -C₆acyloxy, nitro, carboxy, protected carboxy, carbamoyl, carbamoyloxy,cyano, C₁ -C₆ alkylthio, N-(methylsulfonylamino) C₁ -c₆ alkoxy, or othergroups specified. Optionally, the aryl group may be substituted withone, two, or three groups chosen from halogen(especially F), cyano,hydroxy, protected hydroxy, nitro, C₁ -C₆ alkyl, C₁ -C₄ alkoxy, carboxy,protected carboxy, carboxymethyl, protected carboxymethyl,hydroxymethyl, protected hydroxymethyl, aminomethyl, protectedaminomethyl, or an N-(methylsulfonylamino) group. As before, when eitherthe C₁ -C₆ alkyl portion or the aryl portion or both are disubstituted,the substituents can be the same or different.

Examples of the term "substituted C₆ -C₁₀ aryl-C₁ -C₆ alkyl" includegroups such as 2-phenyl-1-chloroethyl, 2-(4-methoxyphenyl)ethyl,2,6-dihydroxy-4-phenyl(n-hexyl), 5-cyano-3-methoxy-2-phenyl(n-pentyl),3-(2,6-dimethyl-phenyl)n-propyl, 4-chloro-3-aminobenzyl,6-(4-methoxyphenyl)-3-carboxy(n-hexyl), 5-(4-aminomethylphenyl)-3-(aminomethyl)(n-pentyl), and the like.

Unless otherwise specified, the terms "heterocycle", "heterocyclicgroup", "heterocyclic" or "heterocyclyl" are used interchangeably hereinand refer to any mono-, bi-, or tricyclic saturated, unsaturated, oraromatic ring having the number of ring atoms designated where at leastone ring is a 5-, 6- or 7-membered hydrocarbon ring containing adesignated number of heteroatoms selected from nitrogen, oxygen, andsulfur, preferably at least one heteroatom is nitrogen (Lang's Handbookof Chemistry, supra). The heterocycle is a 5- or 6-member saturated,unsaturated, or aromatic hydrocarbon ring usually containing 1,2 , or 3heteroatoms, preferably 1 or 2, selected from O, N, and S. Typically,the 5-membered ring has 0 to 2 double bonds and the 6- or 7-memberedring has 0 to 3 double bonds and the nitrogen or sulfur heteroatoms mayoptionally be oxidized, and any nitrogen heteroatom may optionally besubstituted or quarternized. Included in the definition are any bicyclicgroups where any of the above heterocyclic rings are fused to a benzenering. Heterocyclics in which nitrogen is the heteroatom are preferred.

The following ring systems are examples of the heterocyclic (whethersubstituted or unsubstituted) radicals denoted by the term"heterocyclic": thienyl, furyl, pyrrolyl, imidazolyl, pyrazolyl,thiazolyl, isothiazolyl, oxazolyl, isoxazolyl, triazolyl, thiadiazolyl,oxadiazolyl, tetrazolyl, thiatriazolyl, oxatriazolyl, pyridyl,pyrimidyl, pyrazinyl, pyridazinyl, thiazinyl, oxazinyl, triazinyl,thiadiazinyl, oxadiazinyl, dithiazinyl, dioxazinyl, oxathiazinyl,tetrazinyl, thiatriazinyl, oxatriazinyl, dithiadiazinyl, imidazolinyl,dihydropyrimidyl, tetrahydro-pyrimidyl, tetrazolo[1,5-b]pyridazinyl andpurinyl, as well as benzo-fused derivatives, for example benzoxazolyl,benzthiazolyl, benzimidazolyl and indolyl.

Heterocyclic 5-membered ring systems containing a sulfur or oxygen atomand one to three nitrogen atoms are also suitable for use in the instantinvention. Examples of such preferred groups include thiazolyl, inparticular thiazol-2-yl and thiazol-2-yl N-oxide, thiadiazolyl, inparticular 1,3,4-thiadiazol-5-yl and 1,2,4-thiadiazol-5-yl, oxazolyl,preferably oxazol-2-yl, and oxadiazolyl, such as 1,3,4-oxadiazol-5-yl,and 1,2,4-oxadiazol-5-yl. A group of further preferred examples of5-membered ring systems with 2 to 4 nitrogen atoms include imidazolyl,preferably imidazol-2-yl; triazolyl, preferably 1,3,4-triazol-5-yl;1,2,3-triazol-5-yl, 1,2,4-triazol-5-yl, and tetrazolyl, preferably1H-tetrazol-5-yl. A preferred group of examples of benzo-fusedderivatives are benzoxazol-2-yl, benzthiazol-2-yl and benzimidazol-2-yl.

Further suitable specific examples of the above heterocyclic ringsystems are 6-membered ring systems containing one to three nitrogenatoms. Such examples include pyridyl, such as pyrid-2-yl, pyrid-3-yl,and pyrid-4-yl; pyrimidyl, preferably pyrimid-2-yl and pyrimid-4-yl;triazinyl, preferably 1,3,4-triazin-2-yl and 1,3,5-triazin-4-yl;pyridazinyl, in particular pyridazin-3-yl, and pyrazinyl. The pyridineN-oxides and pyridazine N-oxides and the pyridyl, pyrimid-2-yl,pyrimid-4-yl, pyridazinyl and the 1,3,4-triazin-2-yl radicals, are apreferred group. Optionally preferred 6-membered ring heterocycles are;piperazinyl, piperazin-2-yl, piperidyl, piperid-2-yl, piperid-3-yl,piperid-4-yl, morpholino, morpholin-2-yl, and morpholin-3-yl.

An optional group of "heterocyclics" include; 1,3-thiazol-2-yl,4-(carboxymethyl)-5-methyl-1,3-thiazol-2-yl,4-(carboxymethyl)-5-methyl-1,3-thiazol-2-yl sodium salt,1,2,4-thiadiazol-5-yl, 3-methyl-1,2,4-thiadiazol-5-yl,1,3,4-triazol-5-yl, 2-methyl-1,3,4-triazol-5-yl,2-hydroxy-1,3,4-triazol-5-yl, 2-carboxy-4-methyl-1,3,4-triazol-5-ylsodium salt, 2-carboxy-4-methyl-1,3,4-triazol-5-yl, 1,3-oxazol-2-yl,1,3,4-oxadiazol-5-yl, 2-methyl-1,3,4-oxadiazol-5-yl,2-(hydroxymethyl)-1,3,4-oxadiazol-5-yl, 1,2,4-oxadiazol-5-yl,1,3,4-thiadiazol-5-yl, 2-thiol-1,3,4-thiadiazol-5-yl,2-(methylthio)-1,3,4-thiadiazol-5-yl, 2-amino-1,3,4-thiadiazol-5-yl,1H-tetrazol-5-yl, 1-methyl-1H-tetrazol-5-yl,1-(1-(dimethylamino)eth-2-yl)-1H-tetrazol-5-yl,1-(carboxymethyl)-1H-tetrazol-5-yl, 1-(carboxymethyl)-1H-tetrazol-5-ylsodium salt, 1-(methylsulfonic acid)-1H-tetrazol-5-yl, 1-(methylsulfonicacid)-1H-tetrazol-5-yl sodium salt, 2-methyl-1H-tetrazol-5-yl,1,2,3-triazol-5-yl, 1-methyl-1,2,3-triazol-5-yl,2-methyl-1,2,3-triazol-5-yl, 4-methyl-1,2,3-triazol-5-yl, pyrid-2-ylN-oxide, 6-methoxy-2-(n-oxide)-pyridaz-3-yl, 6-hydroxypyridaz-3-yl,1-methylpyrid-2-yl, 1-methylpyrid-4-yl, 2-hydroxypyrimid-4-yl,1,4,5,6-tetrahydro-5,6-dioxo-4-methyl-as-triazin-3-yl,1,4,5,6-tetrahydro-4-(formylmethyl)-5,-dioxo-as-triazin-3-yl,2,5-dihydro-5-oxo-6-hydroxy-as-triazin-3-yl,2,5-dihydro-5-oxo-6-hydroxy-as-triazin-3-yl sodium salt,2,5-dihydro-5-oxo-6-hydroxy-2-methyl-as-triazin-3-yl sodium salt,2,5-dihydro-5-oxo-6-hydroxy-2-methyl-as-triazin-3-yl,2,5-dihydro-5-oxo-6-methoxy-2-methyl-as-triazin-3-yl,2,5-dihydro-5-oxo-as-triazin-3-yl,2,5-dihydro-5-oxo-2-methyl-as-triazan-3-yl,2,5-dihydro-5-oxo-2,6-dimethyl-as-triazin-3-yl,tetrazolo[1,5-b]pyridazin-6-yl and8-aminotetrazolo[1,5-b]-pyridazin-6-yl.

An alternative group of "heterocyclics" includes;4-(carboxymethyl)-5-methyl-1,3-thiazol-2-yl,4-(carboxymethyl)-5-methyl-1,3-thiazol-2-yl sodium salt,1,3,4-triazol-5-yl, 2-methyl-1,3,4-triazol-5-yl, 1H-tetrazol-5-yl,1-methyl-1H-tetrazol-5-yl,1-(1-(dimethylamino)eth-2-yl)-1H-tetrazol-5-yl,1-(carboxymethyl)-1H-tetrazol-5-yl, 1-(carboxymethyl)-1H-tetrazol-5-ylsodium salt, 1-(methylsulfonic acid)-1H-tetrazol-5-yl, 1-(methylsulfonicacid)-1H-tetrazol-5-yl sodium salt, 1,2,3-triazol-5-yl,1,4,5,6-tetrahydro-5,6-dioxo-4-methyl-as-triazin-3-yl,1,4,5,6-tetrahydro-4-(2-formylmethyl)-5,6-dioxo-as-triazin-3-yl,2,5-dihydro-5-oxo-6-hydroxy-2-methyl-as-triazin-3-yl sodium salt,2,5-dihydro-5-oxo-6-hydroxy-2-methyl-as-triazin-3-yl,tetrazolo[1,5-b]pyridazin-6-yl, and8-aminotetrazolo[1,5-b]pyridazin-6-yl.

The terms "heteroaryl group" or "heteroaryl" are used interchangeablyherein and refer to any mono-, bi-, or tricyclic aromatic rings havingthe number of ring atoms designated where at least one ring is a 5-, 6-or 7-membered hydrocarbon ring containing from one to four heteroatomsselected from nitrogen, oxygen, and sulfur, preferably at least oneheteroatom is nitrogen. The aryl portion of the term "heteroaryl" refersto aromaticity, a term known to those skilled in the art and defined ingreater detail in Advanced Organic Chemistry J. March, 3^(rd) ed., pages37-69, John Wiley & Sons, New York (1985).

"Optical isomers", "diastereomers", and "geometric isomers" of some ofthe compounds represented by the formulae described herein arecomprehended to be within the scope of the instant invention, as well asracemic and resolved enantiomerically pure forms and pharmaceuticallyacceptable salts thereof.

"Pharmaceutically acceptable salts" include both acid and base additionsalts.

"Pharmaceutically acceptable acid addition salt" refers to those saltswhich retain the biological effectiveness and properties of the freebases and which are not biologically or otherwise undesirable, formedwith inorganic acids including but not limited to hydrochloric acid,hydrobromic acid, sulfuric acid, sulfamic nitric acid, phosphoric acidand the like, and organic acids such as acetic acid, propionic acid,glycolic acid, pyruvic acid, lactic acid, oxalic acid, maleic acid,malic acid, maloneic acid, succinic acid, fumaric acid, tartaric acid,citric acid, stearic acid, ascorbic acid, benzoic acid, cinnamic acid,mandelic acid, methanesulfonic acid, ethanesulfonic acid, isethionicacid, p-toluenesulfonic acid, salicyclic acid, naturally occurring aminoacids and the like.

"Pharmaceutically acceptable base addition salts" include those derivedfrom inorganic bases such as sodium, potassium, lithium, ammonium,calcium, magnesium, iron, zinc, copper, manganese, aluminum salts andthe like. Particularly preferred are the ammonium, potassium, sodium,calcium and magnesium salts. Salts derived from pharmaceuticallyacceptable organic nontoxic bases includes salts of primary, secondary,and tertiary amines, substituted amines including naturally occurringsubstituted amines, cyclic amines and basic ion exchange resins, such asisopropylamine, trimethylamine, diethylamine, triethylamine,tripropylamine, ethanolamine, 2-diethylaminoethanol, trimethamine,dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine,hydrabamine, choline, betaine, ethylenediamine, glucasamine,methylglucamine, theobromine, purines, piperizine, piperidine,N-ethylpiperidine, polyamine resins and the like. Particularly preferredorganic non-toxic bases are isopropylamine, diethylamine, ethanolamine,trimethamine, dicyclohexylamine, choline, and caffeine.

In general, unless otherwise specified, the abbreviations used for thedesignation of amino acids and the protective groups used therefor arebased on recommendations of the IUPAC-IUB Commission of BiochemicalNomenclature (Biochemistry, 11:1726-1732 (1972). Table 1 provides a listof commonly used symbols or abbreviations (abbr.) used to describe thecompounds of this invention.

                                      TABLE I                                     __________________________________________________________________________    ABBR                                                                              (SMILES) STRUCTURE                                                                           R/S                                                                              COMMON NAME OF COMPOUND                                 __________________________________________________________________________    A   NC(C)C(═O) S  L-Alanine                                                 a NC(C)C(═O) R D-Alanine                                                  Ab NCCCC(═O)  4-aminobutyric acid                                         Ac C(C═O)  Acetyl                                                         Abx NC2(CCCCC2)C(═O)  1-amino-1-cyclohexanecarboxylic acid                Ahp NCCCCCCC(═O)  7-aminoheptanoic acid                                   Ahx NCCCCCC(═O)  6-aminohexanoic acid                                     amb NCc4ccc(cc4)C(═O)  p-aminomethylbenzoic acid                          amF NC(C)(Cc1ccccc1)C(═O) S alpha-methyl-L-Phenylalanine                  apc N3CCC(N)CC3  1-(4-amino-piperidine)                                       api NC3CCNCC3  4-(4-amino-piperidine)                                         Ava NCCCCC(═O)  5-aminovaleric acid                                       B NC(Cc1cc2ccccc2cc1)C(═O) S L-beta-naphthylalanine                       b NC(Cc1cc2ccccc2cc1)C(═O) R D-beta-naphthylalanine                       bA NCCC(═O)  beta-Alanine                                                 bam NCCCCN  1,4-butanediamine                                                 Bmn NC(Cc1cc2ccccc2cc1)CN S L-2-amino-3-(2-naphthyl)propylamine                                    bmn NC(Cc1cc2ccccc2cc1)CN R D-2-amino-3-(2-naphthyl                          )propylamine                                              BOC C(C)(C)OC(═O)  tert-Butoxycarbonyl                                    Bol NC(Cc1cc2ccccc2cc1)CO S L-beta-naphthylalanol                             bol NC(Cc1cc2ccccc2cc1)CO R D-beta-naphthylalanol                             chA NC(CC1CCCCC1)C(═O)  3-cyclohexylalanine                               chf NC(Cc1ccc(C1)cc1)C(═O) R D-4-chloro-Phenylalanine                     cho C4CCC(CC4)C(═O)  cyclohexane carboxylic acid                          cxa NC3CCC(N)CC3  trans-1,4-cyclohexane diamine                               Cxp N4CCN(CC4)C(═O)  1-carboxypiperazine                                  dam N(C)C  N,N-dimethylamine                                                  F NC(Cc1ccccc1)C(═O) S L-Phenylalanine                                    f NC(Cc1ccccc1)C(═O) R D-Phenylalanine                                    fbd N(CCc1ccccc1)CCCCN  N-(2-phenylethyl) butane diamine                      feb N(CCc1ccccc1)CCC(═O)  N-(2-phenylethyl) beta-Alanine                  feg N(CCc1ccccc1)CC(═O)  N-(2-phenylethyl) Glycine                        fem NCCc1ccccc1  1-phenethyl amine                                            G NCC(═O)  Glycine                                                        H NC(Cc1[nH]cnc1)C(═O) S L-Histadine                                      h NC(Cc1[nH]cnc1)C(═O) R D-Histadine                                      hcF NC(Cc1ccccc1)CC(═O) S 3-(S)-benzyl-beta-alanine                       hF NC(CCc1ccccc1)C(═O) S homo-L-Phenylalanine                             hf NC(CCc1ccccc1)C(═O) R homo-D-Phenylalanine                             inip N4CCC(CC4)C(═O)  isonipecotic acid                                   isog N4CC═C(CC4)C(═O)  isoguvacine                                    K NC(CCCCN)C(═O) S L-Lysine                                               k NC(CCCCN)C(═O) R D-Lysine                                               mab N(C)CCCC(═O)  N-methyl-4-aminobutyric acid                            mam NC  methylamine                                                           man NCCc1cc2ccccc2cc1  N-(2-naphthylmethyl)amine                              mBm N(C)C(Cc1cc2ccccc2cc1)CN S N-methyl-[2-amino-3-(2-                           naphthyl)propyl]amine                                                      mbm N(C)C(Cc1cc2ccccc2cc1)CN R N-methyl-[2-amino-3-(2-                           naphthyl)propyl]amine                                                      men N(C)CCc1cc2ccccc2cc1  N-Methyl-2-(2-naphthylethylamine)                   miz N(C)C(Cc1cc2ccccc2cc1)CN(═N) R 2(R)-2-(N-Methylamino)-1-azido-3-                          (2-                                                        N  naphthyl)propane                                                          mor N1CCOCC1  morpholine                                                      N NC(CC(═O)N)C(═O) S L-Asparagine                                     n NC(CC(═O)N)C(═O) R D-Asparagine                                     nba NC(C)(C)CC(═O)  3,3-dimethyl-3-aminopropionic acid                    nbol N(C)C(Cc1cc2ccccc2cc1)CO S N-Methyl-D-beta-naphthylalanol                nL NC(CCCC)C(═O) S L-Norleucine                                           nl NC(CCCC)C(═O) R D-Norleucine                                           nmB N(C)C(Cc1cc2ccccc2cc1)C(═O) S N-Methyl-L-beta-naphthylalanine                              nmb N(C)C(Cc1cc2ccccc2cc1)C(═O) R N-Methyl-D-be                          ta-naphthylalanine                                        nmF N(C)C(Cc1ccccc1)C(═O) S N-Methyl-L-Phenylalanine                      nmK N(C)C(CCCCN)C(═O) S N-Methyl-L-Lysine                                 npA NC(Cc1c2ccccc2ccc1)C(═O) S L-alpha-Naphthylalanine                    npa NC(Cc1c2ccccc2ccc1)C(═O) R D-alpha-Naphthylalanine                    npe NCCc1cc2ccccc2cc1  2-(2-naphthyl)ethylamine                               O NC(CCCN)C(═O) S L-Ornithine                                             o NC(CCCN)C(═O) R D-Ornithine                                             P N1C(CCC1)C(═O) S L-Proline                                              p N1C(CCC1)C(═O) R D-Proline                                              pac n4ccc(cc4)CC(═O)  Pyridine-4-acetic acid                              pam NCCCCCN  1,5-pentanediamine                                               Pg NC(c1ccccc1)C(═O) S L-Phenylglycine                                    pG NC(c1ccccc1)C(═O) R D-Phenylglycine                                    Pol NC(Cc1ccccc1)CO S L-Phenylalanol                                          ppc N1CCC(CC1)C(═O)  Piperidine-4-carboxylic acid                         ppz N3CCNCC3  piperazine                                                      pyc n4ccc(cc4)C(═O)  Pyridine-4-carboxylic acid                           ram NCCCN  1,3-propane diamine                                                S NC(CO)C(═O) S L-Serine                                                  tam NCCN  1,2-ethane diamine                                                  tbm NC(C)(C)C  tert-butylamine                                                tic N7C(Cc8ccccc8C7)C(═O) S D-tetrahydroisoquinoline                      Tic N7C(Cc8ccccc8C7)C(═O) R L-tetrahydroisoquinoline                      W NC(Cc1[nH]c2ccccc2c1)C(═O) S L-Tryptophane                              w NC(Cc1[nH]c2ccccc2c1)C(═O) R D-Tryptophane                              wol NC(Cc1[nH]c2ccccc2c1)CO R D-Tryptophanol                                  Y NC(Cc1ccc(O)cc1)C(═O) S L-Tyrosine                                      y NC(Cc1ccc(O)cc1)C(═O) R D-Tyrosine                                      Y(I) NC(Cc1ccc(O)c(I)c1)C(═O) S 3-Iodo-L-Tyrosine                       __________________________________________________________________________     Notes:                                                                        .sup.a) The above structures are depicted in the SMILES format ("SMILES,      1. Introduction to Encoding Rules" Weinenger, D. J. Chem. Inf. Comput.        Sci. 1988, 28, 31.). They are generally written N to Cterminal with the       points of attachment at the left and/or right hand atoms depending on         sequence position. In cases where attachment would be ambiguous, two          different acronyms are used to depict the two modes, if both are used.        .sup.b) It will be understood that when attached at a terminal position,      the appropriate Cterminal function indicated in the table (i.e. --OH,         --NH2, OMe) is added to complete the structure (acid, amide, Me ester,        respectively). Also, hydrogen atoms are to be added to the terminal amine     functions to fill out the valence.                                       

B. Utility

The compounds of Formula I can be administered to mammals, includingman, to release endogenous growth hormone in vivo. For example, thecompounds can be administered to commercially important mammals such asswine, cattle, sheep and the like to accelerate and increase their rateand extent of growth and the efficiency of their conversion of feed intobody tissue, and to increase milk production in such mammals. Inaddition, these compounds can be administered to humans in vivo as adiagnostic tool to determine whether the pituitary is capable ofreleasing growth hormone. The compounds of Formula I can be administeredin vivo to adults and children to stimulate growth hormone release.

Accordingly, the present invention includes within its scopepharmaceutical compositions comprising, as an active ingredient, atleast one of the compounds of Formula I in association with apharmaceutical carrier or diluent. Optionally, the active ingredient ofpharmaceutical compositions can comprise a growth promoting agent inaddition to at least one of the compounds of Formula I.

Growth promoting agents include but are not limited to; TRH,diethylstilbestrol, theophylline, enkephalins, E series prostaglandins,peptides of the VIP-secretin-glucagon-GRF family and other growthhormone secretagogues such as GHRP-6, GHRP-1 as described in U.S. Pat.No. 4,411,890; benzo fused lactams such as those disclosed in U.S. Pat.No. 5,206,235; and growth hormone releasing hormone (GHRH) and itsanalogs or growth hormone (GH) and its analogs or somatomedins includingIGF-1 and IGF-2 and their analogs.

The compounds of this invention are shown to induce release of growthhormone and IGF-1. It is known to those skilled in the art that thereare many uses for growth hormone and the IGF's. Therefore administrationof the compounds of this invention for purposes of stimulating therelease of endogenous growth hormone or IGF-1 can have the same effectsor uses as growth hormone or the somatomedins themselves. These uses ofgrowth hormone and IGF-1 include the following: stimulating growthhormone release in elderly humans; prevention of catabolic side effectsof glucocorticoids, treatment of osteoporosis, stimulation of the immunesystem, treatment of retardation, acceleration of wound healing,accelerating bone fracture repair, treatment of growth retardation,treating renal failure or insufficiency resulting in growth retardation,treatment of physiological short stature, including growth hormonedeficient children, treating short stature associated with chronicillness, treatment of obesity and growth retardation associated withobesity, treating growth retardation associated with Prader-Willisyndrome and Turner's syndrome; accelerating the recovery and reducinghospitalization of burn patients; treatment of intrauterine growthretardation, skeletal dysplasia, hypercortisolism and Cushings syndrome;Induction of pulsatile growth hormone release; replacement of growthhormone in stressed patients; treatment of osteochondrodysplasias,Noonans syndrome, schizophrenia, depression, Alzheimer's disease,diseases of demeylination, multiple sclerosis, delayed wound healing,and psychosocial deprivation; treatment of pulmonary dysfunction andventilator dependency; attenuation of protein catabolic response after amajor operation; reducing cachexia and protein loss due to chronicillness such as cancer or AIDS; treatment of hyperinsulinemia includingType II diabetes; adjuvant treatment for ovulation induction;stimulating thymic development and prevent the age-related decline ofthymic function; treatment of immunosuppressed patients; treatment ofbone marrow transplanted patients, improvement in muscle strength,mobility, diseases of muscle function, muscular dystrophy's, maintenanceof skin thickness, metabolic homeostasis, enhancing renal function andhemeostasis including acute and chronic renal failure, stimulation ofosteoblasts, bone remodeling, and cartilage growth; stimulation of theimmune system in companion animals; growth promotion in livestockincluding stimulation of milk production in ruminates and wool or hairgrowth.

An alternative use of the GHRP's of this invention, represented byformulae I-V, as well as other GHRP's as defined herein, including butnot limited to GHRP-6 and GHRP-1 as described in U.S. Pat. No.4,411,890; GHRP-2; benzo fused lactam GHRP's such as those disclosed inU.S. Pat. No. 5,206,235; are used in combination with IGF-1 to treatdiseases in which long term IGF-1 treatment is indicated. This use ofGHRP's is to bring serum GH levels back to normal when long-term IGF-1therapy down-regulates the pituitary GH secretion. Such use includes butis not limited to use in the treatment of Type II diabetes.

Other uses of the instant compounds will be apparent from the followingreferences; Amato et al., Journal of Clinical Endocrinology andMetabolism 77(6):1671-1676 (1993), Bengtsson et al., Journal of ClinicalEndocrinology and Metabolism 76(2):309-317 (1993), Binnerts et al.,Clinical Endocrinology 37:79-87 (1992), Bowers, Journal of ClinicalEndocrinology and Metabolism 76(4):817-823 (1993), Cuneo et al., J.Applied Physiol. 70(2):688-694 (1991), Cuneo et al., J Applied Physiol.70(2):695-700 (1991), Degerblad et al., Acta Endocrinologica 126:387-93(1992), Eden et al., Arteriosclerosis and Thrombosis 13(2):296-301(1993), Hartman et al., Horm Res 40:37-47 (1993), Ho et al., Horm Res40:80-86 (1993), Jo/gensen et al., Acta Endocrinologica 125:449-453(1991), Jo/gensen et al., The Lancet June 3:1221-1224 (1989), Lambertset al., Clinical Endocrinology 37:111-115 (1992), McGauley et al., HormRes 33(suppl 4):52-54 (1990), Mo/ller et al., Clinical Endocrinology39:403-408 (1993), O'Halloran et al., Journal of Clinical Endocrinologyand Metabolism 76(5):1344-1348 (1993), Orme et al., ClinicalEndocrinology 37:453-459 (1992), Rodriguez-Arnao et al., Horm Res39:87-88 (1993), Rosen et al., Clinical Endocrinology 40:111-116 (1994),Rosen et al., Acta Endocrinologica 129:195-200 (1993), Rudman et al.,The New England Journal of Medicine 323(1):1-6 (1990), Salomon et al.,The New England Journal of Medicine 321(26):1797-1803 (1989), Shibasakiet al., Journal of Clinical Endocrinology and Metabolism 58(1):212-214(1984), Sonksen et al., Acta Paediatr Scand [Suppl] 379:139-146 (1991),Tauber et al., Journal of Clinical Endocrinology and Metabolism76(5):1135-1139 (1993), Vandeweghe et al., Clinical Endocrinology39:409-415 (1993), Whitehead et al., Clinical Endocrinology 36:45-52(1992), and Bercu et al., U.S. Pat. No. 5,246,920.

Additionally, the most potent compounds of this invention can be used asGH antagonists. It is known that hypothalamic hormones that are superagonists can also be used as antagonists. For example super agonists ofGonadotrophin Releasing Hormone (GnRH) such as GONADORELIN andLEUPROLIDE act either as agonists or antagonists depending on the methodof administration. The actions of the GnRH super agonists are summarizedin Goodman and Gilmans, The Pharmacological Basis of Therapertics, 8thEd., McGraw Hill Inc., p. 1353 (1993). By analogy, it is believed thecontinuous administration of the compounds of formula I-V will lead todown-regulation of the growth response. These molecules can therefore beused as functional antagonists of pituitary GH secretion, therebyantagonizing GH or IGF-1 action.

The uses of such antagonists of GH secretion include but are not limitedto; treatment of excess GH secretion as in acromegaly or gigantism; incancer of the breast, colon and prostate; in diabetes especially in TypeI adolescent patients to counteract the down phenomenon; and in Type Iand Type II patients to directly control blood glucose, and to controlthe long-term affects of diabetes, as for example in retinopathy.

The compounds of this invention can be administered by oral, parenteral(e.g., intramuscular, intraperitoneal, intravenous or subcutaneousinjection or infusion, or implant), nasal, pulmonary, vaginal, rectal,sublingual, or topical routes of administration and can be formulated indosage forms appropriate for each route of administration.

C. Methods of Making

1. General Peptide Synthesis

One method of producing GHRP's involves chemical synthesis of the"polypeptide". This can be accomplished using methodologies well knownto those skilled in the art (see Stewart, J. M. & Young, J. D. SolidPhase Peptide Synthesis Pierce Chemical Co. Rockford, Ill.[1984]; seealso U.S. Pat. No.'s 4,105,603; 3,972,859; 3,842,067; and 3,862,925).

"Polypeptides" of the invention may be conveniently prepared using solidphase peptide synthesis (Merrifield, J. Am. Chem. Soc., 85:2149 [1964];Houghten, Proc. Natl. Acal. Sci USA 82:5132 [1985]). Solid phasesynthesis begins at the carboxy-terminus of the putative peptide bycoupling a protected amino acid to a suitable resin (e.g.chloromethylated polystyrene resin) as shown in FIGS. 1-1 and 1-2, onpages 2 and 4 of Stewart and Young supra. After removal of the α-aminoprotecting group with, for example, trifluoroacetic acid (TFA) inmethylene chloride and neutralizing in, for example TEA, the nextα-amino- and if necessary, side-chain-protected amino acids are added.The remaining α-amino- and, if necessary, side-chain-protected aminoacids are then coupled sequentially in the desired order by condensationto obtain an intermediate compound connected to the resin.Alternatively, some amino acids may be coupled to one another forming apeptide prior to addition of the peptide to the growing solid phasepolypeptide chain.

The condensation between two amino acids, or an amino acid and apeptide, or a peptide and a peptide can be carried out according to theusual condensation methods such as the azide method, mixed acidanhydride method, DCC (N,N'-dicyclohexylcarbodiimide) or DIPC(N,N'-diisopropylcarbodiimide)methods, active ester method(p-nitrophenyl ester method, BOP [benzotriazole-1-yl-oxy-tris(dimethylamino) phosphonium hexafluorophosphate] method,N-hydroxysuccinic acid imido ester method, etc., and Woodward regent Kmethod.

Common to chemical syntheses of peptides is the protection of anyreactive side-chain groups of the amino acids with suitable protectinggroups. Ultimately these protecting groups are removed after the desiredpolypeptide chain has been sequentially assembled. Also common is theprotection of the α-amino group on an amino acid or a fragment whilethat entity reacts at the carboxyl group followed by the selectiveremoval of the α-amino-protecting group to allow subsequent reaction totake place at that location. Accordingly, it is common in polypeptidesynthesis that an intermediate compound is produced which contains eachof the amino acid residues located in the desired sequence in thepeptide chain with various of these residues having side-chainprotecting groups attached. These protecting groups are then commonlyremoved substantially at the same time so as to produce the desiredresultant product following removal from the resin.

Suitable protective groups for protecting the α- and ε- amino side chaingroups are exemplified by benzyloxycarbonyl (CBZ),isonicotinyloxycarbonyl(iNOC), O-chlorobenzyloxycarbonyl (2-Cl-CBZ),p-nitrobenzyloxycarbonyl [Z(NO₂ ], p-methoxybenzyloxycarbonyl [Z(OMe)],t-butoxycarbonyl, (BOC), t-amyloxycarbonyl (AOC), isoborrnyloxycarbonyl,adamatyloxycarbonyl, 2-(4-biphenyl)-2-propyl-oxycarbonyl (BPOC),9-fluorenylmethoxycarbonyl (FMOC), methylsulfonyiethoxycarbonyl (Msc),trifluoroacetyl, phthalyl, formyl, 2-nitrophenylsulphenyl (NPS),diphenylphosphinothioyl (Ppt), dimethylophosphinothioyl (Mpt) and thelike.

Protective groups for the carboxy functional group are exemplified by;benzyl ester (OBzl), cyclohexyl ester (Chx), 4-nitrobenzyl ester (ONb),t-butyl ester (OtBu), 4-pyridylmethyl ester (OPic), and the like. It isoften desirable that specific amino acids such as arginine, cysteine,and serine possessing a functional group other than amino and carboxylgroups are protected by a suitable protective group. For example, theguanidino group of arginine may be protected with nitro,p-toluenesulfonyl, benzyloxycarbonyl, adamantyloxycarbonyl,p-methoxybenzenesulfonyl, 4-methoxy-2,6-dimethylbenzenesulfonyl (Mds),1,3,5-trimethylphenylsulfonyl (Mts), and the like. The thiol group ofcysteine may be protected with p-methoxybenzyl, triphenylmethyl,acetylaminomethyl ethylcarbamoyle, 4-methylbenzyl, 2,4,6-trimethy-benzyl(Tmb) etc., and the hydroxyl group of serine can be protected withbenzyl, t-butyl, acetyl, tetrahydropyranyl and the like.

Stewart and Young supra provides detailed information regardingprocedures for preparing peptides. Protection of α-amino groups isdescribed on pages 14-18, and side-chain blockage is described on pages18-28. A table of protecting groups for amine, hydroxyl and sulfhydrylfunctions is provided on pages 149-151.

After the desired amino acid sequence has been completed, theintermediate peptide is removed from the resin support by treatment witha reagent, such as liquid HF and one or more thio-containing scavengers,which not only cleaves the peptide from the resin, but also cleaves allthe remaining side-chain protecting groups. Following HF cleavage, thepeptide residue is washed with ether, and extracted from the resin bywashing with aqueous acetonitrile and acetic acid.

Preferably in order to avoid alkylation of residues in the polypeptide,(for example, alkylation of methionine, cysteine, and tyrosine residues)a thio-cresol and cresol scavenger mixture is used.

2. Other General Procedures

The peptidomimetic compounds of the invention may also be convenientlyprepared by the methods for peptide synthesis described in monographssuch as ("Principles of Peptide Synthesis, M. Bodanszky, Spring-Verlag,2nd Ed., 1993; "Synthetic Peptides; A Users Guide", G. A. Grant, Ed, W.H. Freeman and Co., 1992; and references sited therein), or by othermethods generally known to one skilled in the art. The synthesis ofcompounds of this invention that are peptidomimetric in nature (i.e.contain other than standard amide bond linkages) may be prepared byextension of the methods described in the specific Examples 1-37 and themethods laid forth in Schemes I-IV below, by the general syntheticmethods described in "Comprehensive Organic Transformations", R. C.Larock, VCH Publishers, 1989, and by methods generally known to oneskilled in the art.

For compounds of claim 1 where the amide linkages (--C(═O)--NH--) arereplaced with amide isostere linkages such as; --CH₂ --NH--, --CH₂--S--, --CH₂ --CH₂ --, --CH═CH-- (cis and trans), --C(═O)--CH₂ --,--CH(OH)--CH₂ --, --CH(CN)--NH--, --O--C(═O)--NH-- and --CH₂ --SO--,amide bond replacing methods known in the art are employed. Thefollowing references describe preparation of amide isostere linkageswhich include these alternative-linking moieties: Spatola, A. F., VegaData 1(3): "Peptide Backbone Modifications" (General Review) (March1983), Spatola, A. F., in "Chemistry and biochemistry of Amino AcidsPeptides and Proteins", B. Weinstein, ed., Marcel Dekker, New York, P.267 (1983); Morley, J. S., Trends Pharm. Sci pp. 463-468; Hudson, D. etal. Int. J. Pept. Prot. Res. 14:177-185 (1979) (--CH₂ NH--, --CH₂ CH₂--); Spatola, A. F., et al., Life Sci. 38:1243-1249 (1986) (--CH₂--S--); Hann, M. M., J. Chem. Soc. Perkin. Trans I 307-314 (1982)(--CH═CH--, cis and trans); Almquist, R. G., et al., J. Med. Chem.23:1392-1398 (1980) (--C(═O)--CH₂ --); Jennings-White C., et al.,Tetrahedron Lett 23:(1982) (--C(═O)--CH₂ --); Szelke, M., et al., EPApplication No. 45665 (1982) Chem Abs:9739405 (1982) (--CH(OH)--CH₂);Holladay, M. W., et al., Tetrahedron Lett 24:4401-4404 (1983)(--C(OH)--CH₂ --); Hruby, V. J. Life Sci 31:189-199 (1982) (--CH₂ S--);and Cho, C. Y. et al, Science 261:1303-1305 (1993) (--O--C(═O)--NH--).

In one embodiment, compounds of the invention are specifically preparedby the methods described in Schemes I-IV. The N-terminal amino group isshown as isonipecotic acid for clarity, but it is understood that thecompounds of this invention with other groups (R^(A)) at this positionare prepared by substitution of the appropriately protected reagent forthe protected isonipecotic acid in the scheme. One may in general use arange of methods for the coupling of the components such as preformedactive esters, acid chlorides, and coupling reagents. For connectionsother than amides, alkylation, acylation, and sulfonylation, forexample, may be accomplished using the appropriately activated reagentand methods described in ("Comprehensive Organic Transformations", R. C.Larock, VCH Publishers, 1989).

3. Specific Schemes

As shown in Scheme I, protected amino acids of the type 1 may bealkylated according to the procedure of Benoitin (Can. J. Chem. 55, 906,1977) to give a variety of N-substituted compounds (2). ##STR29## Toproduce reduced or inverted amide compounds of the Type IIc, (2) may bereduced through the preformed mixed anhydride with sodium borohydride togive protected amino alcohols (3). Conversion of the hydroxyl functionto an amine may be accomplished via Mitsunobu coupling of (3) withhydrazoic acid to give an intermediate protected amino azide. Deblockingof the amino function and coupling to the N-terminal group isconveniently performed at this differentiated stage. In this example,the N-BOC is removed with TFA and the resulting free-based amino azidecoupled to N-BOC-isonipecotic acid using the reagent DCC to give (4).

The intermediate azide (4) can be converted to a variety of compounds ofthis invention. For example, hydrogenation of the azido function givesan amine which can be acylated with a variety of groups, for example,Ar¹ -L² -COCl to give (5) (Scheme I). For the synthesis of the range ofL² 's herein claimed, it is understood that Ar¹ -L² -COCl may besubstituted with a variety of different acylating agents like chlorocarbonates, activated esters, isocyanates, and the like. For example,2-naphthoylchloride, benzylchloroformate, phenylacetyl chloride,dihydrocinnamoyl chloride, and phenylisocyanate may be used to givecompounds (6) with a range of linkers L². Global deprotection then gives6, for R^(C) =H. Incorporation of the N-substitution R^(C), into (5) canbe accomplished via alkylation of (5), for example via deprotonationwith sodium hydride and reaction with an alkyl halide. Deprotectiongives (6) (R^(C) ≢H).

For synthesis of N-sulfonamido compounds, the amine produced viareduction of (4) can be sulfonylated, alternately alkylated at nitrogen(for R^(C) ≢H), and deprotected to give (8).

For the synthesis of compounds (6) and (8) where R^(B) =H and R^(C) ≢H,it may be more convenient to incorporate the substituent R^(C) viareductive amination. For example, using one equivalent of an appropriatealdehyde and sodium cyanoborohydride, R^(C) can be introduced into theamine from reduction of (4), prior to acylation or sulfonylation to (6)or (8), respectively.

Compounds IIc in which X=H, alkyl, substituted alkyl, and the like, maybe synthesized via the route shown in Scheme II. ##STR30## Theintermediate protected amino acids (1) or (2) (from Scheme I) areconverted to protected amino aldehydes (9), conveniently via DIBALreduction of the derived N-methyl-N-methoxy-amide. Subsequent reductiveamination with an appropriately substituted amine gives (10).Alternatively, (10) can be prepared from (3) via conversion of thealcohol to a tosylate or other suitable leaving group, and displacementwith an appropriately substituted amine (Scheme II). It should beapparent that a wide variety of amines could be used in these two routesto (10) including tryptamine, N-methyl-(2-naphthyl)ethyl amine,alpha-methylphenethylamine, tryptophanol, andN-methyl-beta-naphthylalanol. The amine may also be part of aheterocycle, i.e. 2-benzyl- or naphthylmethyl-piperidine.

This reductive amination strategy is a general method for incorporationof a reduced amide isostere into a polyamide chain. Thus, substitutionof an appropriately protected amino acid derivative or peptide with afree alpha-amine, for the amine component in Scheme II (9) to (10)provides a protected, reduced amide isostere, intermediate. In thismanner, compounds IIIb-IIIe may be prepared using appropriate orthogonalprotecting groups for the reactive functionality. This method may alsobe employed when the amine component is attached to a solid support,suitable for peptide synthesis, providing a convenient method for thesynthesis of longer peptidic compounds.

Completion of the synthesis of compounds of the type 11 (Scheme II) (andby analogy, compounds IIIb-IIIe), requires deprotection of the aminogroup and coupling to an appropriately protected N-terminal moiety,shown in Scheme II as N-R¹ -isonipecotic acid for clarity. Depending onthe particular substituents X, R^(B), and R^(C) in (10), it may benecessary to orthogonally protect reactive functionality prior toremoval of the N-terminal blocking group. For example, for (10) (R^(C)=X=H) the secondary amine in (10) can be acylated with FMOC-Cl (e.g.R^(C) =FMOC) prior to removal of the N-terminal BOC. This ensures thatthe subsequent acylation occurs only at the terminal amine.

It will be noted that a wide variety of N-terminal groups can beattached to the intermediate deprotected (10). Any suitably protectedamino acid, i.e. BOC-4-aminobutyric acid, N-alkyl-isonipecotic acid, maybe attached using a standard coupling reagent. Also, protected activeesters, anhydrides, and acid chlorides, may be used. For the synthesisof urea type linkages, the intermediate deprotected (10) may be reactedwith carbonyl diimidazole or phosgene, followed by addition of asuitably protected or symmetrical amine. In particular, reaction withpiperazine, propane diamine, or N1,N4-dimethylpropanediamine, givepreferred compounds.

For the synthesis of compounds with an N-terminal carbamate linkage, theintermediate deprotected (10) (R^(C) ≢H) may be reacted with carbonyldiimidazole or phosgene, followed by addition of a suitably N-protectedamino alcohol such as BOC-aminoethanol, BOC-aminopropanol, and BOC-2- or3-hydroxypiperidine. Alternatively, the intermediate deprotected (10)can be reacted directly with a preformed N-blocked-chloroformate.

The final step necessary for completion of the synthesis of thecompounds of type 11 is removal of the protecting functionality usingappropriate conditions (for a general monograph on protecting groups,see Greene, W. T., Wuts P. G. M. Protective Groups in Organic Synthesis,2nd Ed., John Wiley & Sons, NY [1991]).

It will be noted that these methods for incorporation of differentN-terminal groups (e.g. R^(A) 's) are generally applicable to thecompounds of this invention and not limited to the particular example ofScheme II.

The synthesis of the peptidomimetic compounds IId-IIg, are shown belowin Schemes III-VI. For the synthesis of IId (Scheme III), protectedamino acid (2) or (1 for R^(B) =H) is converted to the homologous methylester via rearangement of the diazoketone with Ag(I) in methanol.##STR31## Reduction of the ester provides alcohol (12) which, whenconverted to a tosylate or similar leaving group, can be displaced by alarge range of substituted amines, as exemplified by the conversion of 3to 10 (Scheme II). Deprotection of the product (13) and acylationprovides (14) after deprotection.

Compounds of the type IIe can be prepared as shown in Scheme IV.##STR32## Substituted amine (15) is acylated with bromoacetyl bromide togive (16), which is reacted with a second amine to give (17). Acylationwith an appropriate N-terminal moiety gives (20). For example, aprefered N-terminus, 4-carboxymethylpiperidine (19), is prepared viahomologation of BOC-isonipecotic acid (18), and acylated onto (17) withDCC. Deprotection and optional alkylation of the terminal amine provides(20). Reductive amination with an appropriate aldehyde is an alternatemethod for the incorporation of R¹ substituents onto the terminal amine.This is a generally applicable method, useful for many compounds of thisinvention.

Compounds of the type IIf (Scheme V) can be prepared from (17) via LAHreduction of the amide functionality. Acylation with (19), deprotection,and optional N-alkylation provides (21). ##STR33## The pseudosymmetrical compounds of type IIg are preparable via the route shown inScheme VI. Conversion of arylamine (22) to (23) is analogous to thepreparation of (17) above. ##STR34## LAH reduction provides asymmetrical or unsymmetrical substituted ethane diamine which isacylated simultaneously at both nitrogens with 19 or another appropriatereagent. Deblocking as above gives (24).

D. Preferred Embodiments

The present invention is based on the discovery of several new classesof small peptidomimetics that cause the release of growth hormone inmammals. It is a preferable object of the present invention to provideagents that are selective for GH release and have suitable safety andefficacy for chronic administration to mammals. In a more preferedembodiment, the present invention provides compounds which are suitablefor oral, intranasal, or pulmonary delivery. It is an aim of the mostprefered embodiments of the present invention to provide compounds thatare superior to the prior art by the above criteria. It is furtherprefered that the compounds be readily synthesizable in optically pureform where necessary.

In view of the foregoing, the prefered compounds of this invention havean EC₅₀ in the rat "pit" cell assay of less than about 1.0 nM and mostpreferably less than about 0.5 nM. Prefered compounds of this inventionalso have a molecular weight less than 650 da and most preferably lessthan 600 da. Prefered embodiments of the compounds of this invention arerepresented by structural Formula (I) ##STR35## where the symbols offormula (I) are selected from the following:

From the substructures shown for group A of Formula (I), those preferedA's are selected from; ##STR36## while the most preferred A'sincorporate either an amide or carbamate linkage as in; ##STR37##

With respect to certain combinations of L¹ -Ar¹ and B, it is prefered touse a commercially available amino acid as a starting material.

For the groups R^(A), of substructure A, prefered embodimentsincorporate functionality that places a basic nitrogen atom (or prodrugform thereof) at a distance of approximately 4 to 8 C--C bonds from theattachment point of L¹ -Ar¹, in a through-bond measurement. For example,preferred R^(A) 's, of the most prefered A substructures above, includealkyl amines (CH₂)_(n) NR² R³ (where n=2 to 4) and saturatedsix-membered ring heterocycles containing 1 or 2 nitrogen atoms, for theamide-linked A's, and (CH₂)_(n) NR² R³ (where n=2 or 3) and 3- or4-substituted saturated six-membered ring heterocycles containing 1 or 2nitrogen atoms, for the carbamate-linked A's. Prefered R's attached atthe nitrogen atom of R^(A) include hydrogen, methyl, ethyl,2-hydroxyethyl, and 2-hydroxypropyl. More prefered, are those R^(A) 'sthat place the amine at approximately 6 C--C bonds from the attachmentpoint of L¹ -Ar¹, measured in a through-bond manner, as is the case withthe most prefered R^(A) 's (CH₂)₃ NR² R³, 4-piperidinyl, andpiperazinyl, for the amide linked A's, and (CH₂)₃ NR² R³ for thecarbamate linked A's, where R² and R³ are chosen from the group hydrogenand methyl. In the case of the carbamate where R^(A) is attacheddirectly to nitrogen, an additionally prefered R^(A) forms a piperazine,where the carbamate nitrogen is incorporated as a ring atom. Thus, amost prefered embodiment of the present invention incorporates asubstructure A of the following composition: ##STR38## where the groupsR^(B) are hydrogen or lower alkyl.

In the most prefered embodiment of the instant invention, the Asubstructure of Formula (I) is an amide derived from attachment ofisonipecotic (inip) acid (piperidine-4-carboxylic acid) ##STR39## wherethe groups R^(B) and R¹ are hydrogen or lower alkyl.

As is taught in the present invention, the above substructure A'sdisplay the amine functionality at a near optimal distance from L¹ -Ar¹in Formula (I) and thus it is prefered that the appended R^(A) 's forother substructure A's of the instant invention mimic this distance asclosely as possible, preferably through the incorporation of arigidifying carbo- or heterocyclic substructure.

Prefered embodiments of the other substructures of the compoundrepresented in Formula (I) are as follows:

For the groups a and b, hydrogen and methyl, independently selected, aremore prefered. In a most preferred embodiment a and b are both hydrogen.

For the substructure B, which links the two aromatic sidechains, theamide, amine, and ether of the following functions are more prefered;##STR40##

The most preferred B's are selected from: ##STR41## where R^(C) isadditionally preferred to be hydrogen or methyl.

Moreover, when the substructure A, B, and/or C are comprised of theamide function C(═O)NH, the present invention teaches that the NH isreplacable by MNe with retention of biological activity. It is thereforea most prefered embodiment of the present invention that the groupsR^(B), R^(C), and R^(D) be independently selected from methyl andhydrogen, a particular combination chosen so as to optimize for desiredproperties of the molecule, such as stability and lipophilicity.

From the prefered list of substructures C, of Formula I, the moreprefered embodiments are conveniently discussed by class. For the"pentapeptide, short series", exemplified by the most prefered(inip)bwFK-NH₂ (where C is --C(═O)--Phe--Lys-amide), and Formula Iabelow, prefered embodiments include, in addition to the most prefered--C(═O)--Phe--Lys-amide, the substitution for Lys (Y) by the n-alkyldiamines H₂ N(CH₂)_(n) NH₂, where n=2-6, and amino amides selected fromthe common amino acids. ##STR42## As is taught in the present invention,a wide range of substitution is allowable at the Lys (Y) and, to alesser extent, the Phe (X) position. It is therefore preferable toselect from all possible C-terminal groups, those that are inexpensive,and improve the overall physical properties of the compound. ##STR43##

For the Phe (X) position in the above formula (Ib) and the"tetrapeptide, short series", the Phe is most prefered when a C-terminalamide is included, as are L-alpha-naphthylalanine,L-beta-naphthylalanine, and Tyr. In the short series, exemplified by(inip)bbF-NH₂, the C-terminal carboxamide is a prefered embodiment. Alsoprefered are the amides N,N-dimethyl, N-methyl and morpholinyl. In afurther prefered embodiment, the carboxamide is replaced with the freeacid and the reduced congener CH₂ OH and hydrogen.

An additional class of most prefered compounds (Formula Ic) are obtainedby the replacement of the Phe in the above structures with anon-aromatic residue. Most prefered among this class are the compoundswhere X (below) is an amide derived from the lower alkyl diamines andthe lower alkyl aminocarboxamides. Most preferable is when X is butanediamine. ##STR44## Most preferable is a compound where X is butanediamine and B is a N-methyl amide. Further most preferable compoundsinclude those where X is NH₂, alkyl amides therefrom, OH, and it's loweralkyl esters.

In the "micro series", exemplified by (inip)b(wol), and depicted below,Z is prefered to be CH₂ OH, CH₂ OC(═O)R², CH₂ NR² R³, CH₂ OR, andhydrogen. Most prefered is Z═CH₂ OH or hydrogen. ##STR45## From theprefered list of L¹ -Ar¹ 's and L² -Ar² 's detailed in claim 1, the mostprefered are chosen from CH₂ Ar, where Ar is preferably 1- or2-naphthyl, 3-indoyl, or substituted phenyl. In a most highly preferedembodiment, L¹ -Ar¹ is CH₂ (2-naphthyl) and L² -Ar² is CH₂ (3-indoyl) orCH₂ (2-naphthyl).

Other most prefered compounds of the present invention include:##STR46## E. Biological Activity

1. In Vitro Activity

A. In Vitro EC₅₀

The "pit" EC₅₀ values for all GHRPs were determined by the GHdose-response to the GHRP using the rat pituitary monolayer culturesystem detailed in Example 38. The results are provided in Tables II-VIin Example 39. Table II details selected biological data for prior artcompounds including GHRP-6 with a "pit cell" EC₅₀ of 6.2±1.5 nM (n=5).Table III details selected biological data from 64 compounds fromformula IV. Included in this novel class of compound, which issignificantly smaller than GHRP-6, is (inip)-bbFK-NH₂ with an EC₅₀ of0.18±0.04, over 30fold more potent than GHRP-6. Table IV detailsselected biological data from 63 compounds derived from formula III,including (inip)bb(feg) with an EC₅₀ of 0.25±0.19 (n=3); almost 25-foldmore potent than GHRP-6. Table V details selected biological data from23 compounds from formula II including (inip)b(wol) (EC₅₀ =10.6±6.2;n=3) with a EC50 roughly equivalent to GHRP-6. Table VI details selectedbiological data from "retroinverso" compounds including the most potent,(Ab)bBB(ram), with an EC₅₀ of 2 nM (n=2).

B. In Vitro Characterization

In addition, representatives from novel classes of GHRP were furthercharacterized in vitro to determine whether these compounds were actingin a manner analogous to "GHRP-6". The representatives include: fromformula IV (inip)-bbFK-NH₂, from formula III (inip)bb(feg) and fromformula II (inip)b(wol). The characterization results are detailedbelow. All experiments had a minimum of three replicates.

1. Representative from Formula IV ##STR47##

A representative dose response for GH release in the rat "pit" cellassay over a 15 min. exposure to increasing concentrations of(inip)-bbFK-NH₂ is demonstrated in FIG. 2 (see Example 38 for all assayprotocols). GH release is significantly (P<0.05) increased at 0.3 nM andreaches a plateau by 1 nM with an EC₅₀ of 0.16 nM. The mean (n=3) EC₅₀for (inip)-bbFK-NH₂ was 0.18±0.04 nM, over 30-fold more potent than(GHRP-6) (6.2±1.5 nM; n=5).

To demonstrate that the novel (inip)-bbFK-NH₂ was acting in a manneranalogous to "GHRP-6", challenges were carried out using combinations of(GHRP-6), (inip)-bbFK-NH₂ and GHRH (see FIG. 3). GHRP-6 and(inip)-bbFK-NH₂ (100 nM) both caused 3-fold increases in GH levels, butno additional increase was observed when these two GHRP's were used incombination. This suggests that both GHRP's may act at the same site. Incontrast, both GHRP's showed synergy with GHRH in the rat "pit" cellassay indicating that neither GHRP acts via the GHRH receptor.

A desensitization effect on the putative GHRP receptor was observed whencells were sequentially challenged with fresh (inip)-bbFK-NH₂ every 15min. (FIG. 4). The GH release was decreased after the second 15 min.incubation (total 30 min. exposure to (inip)-bbFK-NH₂) and nosignificant GH release compared to control occurred during the final(inip)-bbFK-NH₂ challenge. However when GHRH (10 nM) was added to thenext 15 min. incubation, a significant GH response occurred, consistentwith the model of separate receptors for GHRH and the GHRP's.

Somatostatin is known to suppress GHRP-stimulated GH release. At 1, 10and 100 nM (inip)-bbFK-NH₂ significant elevations of GH were observed.Somatostatin (20 nM) coincubation with (inip)-bbFK-NH₂ at the sameconcentrations suppressed this enhanced release (FIG. 5).

Other evidence that (inip)-bbFK-NH₂ evokes the GHRP receptor includesthe response to the GHRP receptor antagonist HwkWfK. As demonstrated inFIG. 6, while high doses (10 μM) were required, HwkWfK did antagonize(inip)-bbFK-NH₂.

The specificity of (inip)-bbFK-NH₂ in vitro was demonstrated in that LH,FSH, TSH or ACTH release was unchanged by 100 nM (inip)-bbFK-NH₂.Prolactin concentrations were significantly increased but less than 2fold (FIGS. 7-9).

Ca⁺⁺ flux determinations are shown in FIG. 10. The left panels (A and C)show the basal Ca⁺⁺ pattern immediately prior to addition of vehicle or(inip)-bbFK-NH₂. Twenty-one (21) seconds after (inip)-bbFK-NH₂application the increased Ca flux in panel D is demonstrated by lightercolors in some of the cells of this heterologous population. As acontrol, addition of vehicle did not alter the Ca⁺⁺ profile (B), butaddition of the calcium secretagogue ionomycin resulted in maximalstimulation of all the cells (data not shown).

2. Representative from Formula III ##STR48##

This novel GHRP is smaller by about one (lysine) amino acid residuecompared to (inip)-bbFK-NH₂ described above. Dose dependent GH releasewith (inip)bb(feg) is shown in FIG. 11. GH release by rat pituitarycells to increasing concentrations of (inip)bb(feg) (left panel) over a15 minute incubation shows a dose dependent increase. The right panelshows data points and curve used to calculate the EC₅₀ of 0.09 nM forthe GHRP (inip)bb(feg). The mean EC₅₀ (n=3) for (inip)bb(feg) was0.25±0.19 nM.

To demonstrate that (inip)bb(feg) also acts at the proposed "GHRPreceptor", GH response to GHRP-6 (100 nM) and (inip)bb(feg) (100 nM) wasmeasured as shown in FIG. 12. The combination of these two GHRP'sproduced a GH release significantly greater than the control but GHrelease was not synergistic when these GHRP's were added in combination.GHRH (100 nM) elicited a mild GH response which was synergistic incombination with either GHRP-6 or (inip)bb(feg) indicating these GHRP'sevoke a receptor different from GHRH.

Somatostatin suppression of (inip)bb(feg)-stimulated GH release is shownin FIG. 13. GH release with 100 nM (inip)bb(feg) was totally suppressedin the presence of 20 nM somatostatin consistent with the SS suppressionresponse of other GHRP's.

Similarly, desensitization of the "GHRP receptor" upon challenging ratpituitary cells with three sequential 15 min. incubations with fresh(inip)bb(feg) is demonstrated in FIG. 14. GH release from the samepituitary cells over three sequential 15 minute incubations with(inip)bb(feg) (100 nM) demonstrated a classic GHRP desensitizationpattern. After a total of 45 minutes, GH release was markedly decreasedin response to (inip)bb(feg) but these cells were able to release moreGH in response to a final 15 minute incubation with GHRH (10 nM).

3. Representative from Formula II ##STR49##

This novel GHRP is still smaller than (inip)bb(feg), containing only twoaromatic residues (b-wol) compared to the three for (inip)-bbFK-NH₂ and(inip)bb(feg). A dose dependent GH release with (inip)b(wol) isdemonstrated in FIG. 15. GH release by rat pituitary cells to increasingconcentrations of (inip)b(wol) (left panel) over a 15 minute incubationshows a dose dependent increase. The right panel shows the data pointsand curve used to calculate the EC₅₀ of 3.9 nM for the GHRP(inip)b(wol). The mean EC₅₀ (n=3) for (inip)b(wol) was 10.6±6.2 nM.

Again, to demonstrate that (inip)b(wol) acts at the proposed "GHRPreceptor", GH response to GHRP-6 (100 nM) and (inip)b(wol) (100 nM) wasmeasured as shown in FIG. 16. GH release with there two GHRP's wassignificantly greater than control but not synergistic. In contrast,GHRH (100 nM) elicited a mild GH response which was synergistic incombination with either GHRP-6 or (inip)b(wol).

Somatostatin suppression of (inip)b(wol)-stimulated GH release isdemonstrated in FIG. 17. GH release to 100 nM (inip)b(wol) was totallysuppressed in the presence of 20 nM somatostatin consistent with the SSsuppression response of other GHRP's.

Finally, the desensitization effect of the "GHRP receptor" uponchallenging rat pituitary cells with three sequential 15 min.incubations with fresh (inip)b(wol) is shown in FIG. 18. GH release fromthe same pituitary cells over three sequential 15 minute incubationswith (inip)b(wol) (100 nM) demonstrated a classic GHRP desensitizationpattern. After a total of 45 minutes, no significant release of GH wasobserved in response to (inip)b(wol) but these cells were able torelease GH in response to a final 15 minute incubation with GHRH (10 nM)

4. Summary of In Vitro Characterization

Clearly, by the functional assays demonstrated herein, representativesfrom each novel class of compound (Formulas II, III, IV and V) elicit GHrelease in a manner analogous to "GHRP-6". All these classes ofcompounds released GH in a dose-dependent manner, were synergized byGHRH but not GHRP-6, and had receptor desensitization after continuousexposure to the GHRP while maintaining the ability to respond to GHRH;all consistent with these compounds working through the putative "GHRPreceptor". These assays are well-accepted and have been used extensivelyin the literature. (Cheng et al., Endocrinology 132:2727-2731 [1993];Blake and Smith, Journal of Endocrinology 129:11-19 [1991]; Cheng etal., Endocrinology 124:2791-2798 [1989]; Smith Science 260:1640-1643[1993]; and Akman et al., Endocrinology 132:1286-1291 [1993]).

Additionally, a representative from Formula IV, (inip)bbFK-NH₂, wasfurther characterized and demonstrated a selective GH release fromheterogeneous pituitary cells (excepting a mild, but significantincrease in prolactin), ability to release GH via a Ca⁺⁺ flux mechanism,and to be inhibited by a GHRP antagonist. (Bowers et al., Endocrinology128:2027-2035 [1991]).

These data are consistent with the view that all four novel classes ofGHRP's elicit GH release in a manner analogous to "GHRP-6", and thus mayopperate via the same mechanism to release GH in vitro.

2. In Vivo Activity in Normal Rats

To determine if the new GHRP molecules showed efficacy in vivo, young(90 day old) and adult (120 day old) rats were treated with the GHRP'sof this invention and GHRH according to the protocols in Examples 41 and44. Rat GHRH, which has been shown to increase body weight in normalyoung female rats was used as a positive control in these experiments(Clark and Robinson, Nature 314:281-283 [1985]).

A. Body Weight Gain in Normal Rats

Body weight gains plotted against time for the 5 treatment groups areshown in FIG. 19. Both (inip)bbFK-NH₂ and rat GHRH induced significantbody weight gain compared to the vehicle excipient rats. By Day 5 oftreatment the weight gains of all the treated groups were statisticallysignificantly greater than the excipient treated rats. The dose-relatednature of the body weight gains to (inip)bbFK-NH₂ is presented in FIG.19. The weight gain in response to the much larger dose of rat GHRH (600μg/day) was very similar to that induced by the medium dose (20 μg/day)of (inip)bbFK-NH₂. In addition, as little as 4 μg/day (equal to 166ng/hr) of (inip)bbFK-NH₂ induced a significant weight gain in youngnormal rats. The great potency of (inip)bbFK-NH₂ can be seen from thiscomparison with GHRH and from the very low dose required to induce asignificant anabolic effect in vivo.

Body weight gains plotted against time for the groups of normal adultfemale rats treated with other GH secretagogues are shown in FIG. 26.The groups treated with GHRH and GHRPs 6, (inip)bbF-NH₂, and(inip)b(nmb)(bam) showed significant body weight gain but the grouptreated with L-692,585 showed no significant weight gain. Although theresponse to GHRP (inip)bbF-NH₂ appears to be larger than that to theother secretagogues, this difference was not statistically different.The weight gain in response to GHRP (inip)bbF-NH₂ (36±8 g in 7 days) wassimilar to that to GHRP (inip)bbFK-NH₂ (36±3 g in 14 days) shown in FIG.20.

These studies show the various classes of GH secretagogues of thisinvention have significant anabolic effects in normal rats with intactpituitary function. One prior art molecule with minimal activity wasL-692,585 a molecule of relatively low potency both in vitro and invivo. It is believed, however, that if a larger amount of this moleculehad been given significant anabolic effects would have resulted.

B. Organ Weight Gain in Young Normal Rats

Organs were weighed at sacrifice in these experiments to measure theeffects of these treatments on the major organ systems. The pituitaryand the kidney weight were not affected by treatment. Spleen weight wasincreased by high dose (inip)bbFK-NH₂ (100 μg/day) and by GHRH(excipient 532±25 mg; high dose (inip)bbFK-NH₂, 628±26 mg; GHRH 624±23mg). Heart weight was increased by GHRH treatment (p<0.05) and tended tobe increased by high dose (inip)bbFK-NH₂ (p<0.10 but >0.05) compared toexcipient treated controls. The thymus was also increased in weight byboth (inip)bbFK-NH₂ and GHRH. Thymus weight in excipient treated ratswas 485±21 mg, 584±34 mg in high dose (inip)bbFK-NH₂ treated rats, and575±39 mg in GHRH treated rats. The liver increased in weight in adose-dependent manner with (inip)bbFK-NH₂. The weight of the liver inthe excipient treated rats was 8.75±0.24 g, and with increasing doses of(inip)bbFK-NH₂ liver weight increased from 9.40±0.37 g to 9.70±0.29 gand 10.14±0.29 g for low medium and high doses of (inip)bbFK-NH₂,respectively. Liver weight was also significantly increased by treatmentwith GHRH. In these experiments there was no statistically significantincrease in epiphyseal plate width with either (inip)bbFK-NH₂ or GHRHtreatment.

C. Organ and Body Weight Gain Summary

These experiments shows that (inip)bbFK-NH₂ has a range of anaboliceffects in normal young female rats. This anabolic effect was seen byincreases in body weight, liver weight, spleen weight and thymus weight,with a tendency for heart weight to also increase compared to excipienttreated control rats. The effect of the GHRP (inip)bbFK-NH₂ was alsodose related with doses of 4, 20 and 100 μg/rat/day all being effectiveanabolic doses. In these experiments the two highest doses of(inip)bbFK-NH₂ had equivalent effects. Therefore as little as 166 ng/hrof (inip)bbFK-NH₂ (for 200 g rats; 0.83 μg/kg/hr) was effective atinducing an anabolic effect.

The dose-related effect of (inip)bbFK-NH₂ on the liver is a goodindicator of the amount of GH secretion caused by the GHRP(inip)bbFK-NH₂. Liver growth is particularly sensitive to stimulation byGH, and the increased liver weight is the expected response to anincreased secretion of GH caused by both (inip)bbFK-NH₂ and GHRH. Kidneyshows a relatively poor growth response to GH treatment; the lack of aneffect of (inip)bbFK-NH₂ on this organ is therefore the expected result.Wagner and Scow, Endocrinology 61:419-425 (1957); Clark et al.,Endocrinology and Metabolism 1:49-54 (1994).

The effects of (inip)bbFK-NH₂ on the weight of the thymus and spleenindicate that the instant novel GHRPs would be expected to stimulateimmune function. Other studies have shown that GH and IGF-1 cansignificantly stimulate immune function, so it would be expected thatGHRPs of this invention, by increasing GH secretion, would alsostimulate immune function (Kelley, Ann, N.Y. Acad. Sci. 594:95-118(1990), Clark et al., J. Clin. Invest 92:540-548 (1993).

(inip)bbFK-NH₂ tended to increase cardiac weight, indicating asignificant anabolic effect of this GHRP on the heart. GH and IGF-1 havebeen shown to be efficacious in animal models of congestive heartfailure, and there is data that GH is effective in humans at improvingcardiac function in growth hormone deficient adults. This data suggeststhat (inip)bbFK-NH₂ would also be effective at improving cardiacfunction and in the treatment of cardiac congestive heart failure (saccaet al., Endocrine Reviews 15:555-573 [1994]).

The GHRP (inip)bbFK-NH₂ was also effective at stimulating an anabolicresponse when delivered by continuous infusion. The delivery of(inip)bbFK-NH₂ by SC infusion is an effective treatment and a similareffect could be achieved by any method that maintained a near continuousexposure of the instant GHRP's. For example, oral delivery, transdermalpatch, or other delivery systems designed to maintain a continuousexposure to these GHRP's would be appropriate.

D. Comparison of GHRP Infusion Versus Injections in Normal Rats

Body Weight Gain: The GHRP (inip)bbFK-NH₂ at 20 and 100 μg/day,delivered by both injection and infusion, induced significant bodyweight gain compared to excipient treated rats (see Example 42). By Day2 of treatment, the weight gains of all the treated groups werestatistically significantly greater than the excipient treated rats. Thedose-related nature of the body weight gains to injections of(inip)bbFK-NH₂ can be seen in FIG. 20. In contrast there were similarweight gains in response to infusions of 20 and 100 μg/day of(inip)bbFK-NH₂. In addition, there were very different patterns ofweight gain in response to infusions or injections of (inip)bbFK-NH₂ ascan be seen in FIG. 21.

FIG. 21 also shows an initial rapid weight gain over 2 days in responseto the high dose of infused (inip)bbFK-NH₂ (100 μg/d), followed by amarked absence of any further response for the duration of theexperiment. This provides evidence that continuous exposure to highdoses of GHRPs can induce tachyphalaxis and thus function as GHantagonists.

In contrast to infusions, periodic injections of (inip)bbFK-NH₂maintained a significant growth response. Twice daily injections of 10μg of the GHRP (inip)bbFK-NH₂ produced a large (30 gram) weight gain inadult female rats.

Organ Weights: Organ weights at sacrifice were measured to determine theeffects of injection verses infusion of (inip)bbFK-NH₂ on major organsystems. The eviscerated and skinned carcass was significantly heavierin the high dose (152.4±2.5 g) and low dose (152.8±2.8 g) (inip)bbFK-NH₂injected animals compared to controls (141.8±3 g). The skin was heavierin animals injected with (inip)bbFK-NH₂ and in those infused with lowdose (inip)bbFK-NH₂ compared to control animals. When the skin andcarcass weight were expressed as a percentage of body weight there wasno significant effect due to treatment, indicating that the weight gainwas due to a proportional increase in the whole body size of the rats.Soleus muscle, kidney, and liver were also unaffected by treatment.Injections of high dose (inip)bbFK-NH₂ significantly increased heartweight compared to controls (1.15±0.10 g vs. 0.96±0.03 g). Thymus weightwas increased by high dose injections of (inip)bbFK-NH₂ (0.33±0.02 g)compared to high dose infusion animals (0.25±0.03 g). Epiphyseal platewidth was increased by high dose (inip)bbFK-NH₂ injections (191±8 μm)compared to low dose injections (160±11 μm) whereas (inip)bbFK-NH₂infusions did not significantly increase cartilage growth. Serum IGF-1concentrations were not significantly affected by either mode ofdelivering (inip)bbFK-NH₂.

Serum chemistries were measured in the blood samples obtained atsacrifice. Enzyme levels indicative of cardiac, liver, muscle and kidneyfunction were measured. there were no statistically significant effectsof (inip)bbFK-NH₂. In addition, metabolites (glucose, blood ureanitrogen, creatinine, total protein, albumen, cholesterol, bilirubin)and ions (calcium, phosphate, sodium, potassium and chloride) weremeasured. The only metabolite showing some evidence of changing was theserum triglyceride. There was some evidence that serum triglyceride wasincreased by high dose injections of the GHRP (inip)bbFK-NH₂ but not bylow dose injections (control 126±11 mg %, high dose 183±17 mg %, lowdose 128±11 mg %) although this effect failed to reach statisticalsignificance.

This experiment clearly shows that the GHRP (inip)bbFK-NH₂ when given byeither injections or infusions has a range of anabolic effects in normaladult female rats. This growth promoting effect was seen by increases inbody weight gain, carcass weight, skin weight, heart weight and thymusweight compared to excipient treated control rats. In this experimenttwo injections per day of 10 μg of (inip)bbFK-NH₂ caused a weight gainnearly equal to that of two injections per day of 50 μg of(inip)bbFK-NH₂. Therefore 10 μg of (inip)bbFK-NH₂ (for 200 g rats; 50μg/kg/day) appears to be a maximal dose of (inip)bbFK-NH₂ for inducingan anabolic response. Acute intravenous injection experiments with(inip)bbFK-NH₂ in 80 gram rats (Example 40) over a 25-fold range ofdoses (e.g. 1.0, 0.2, and 0.04 μg/injection) demonstrates that inductionof GH secretion occurs within this dose-response range (ED₅₀ 0.2μg/rat). In 200 g rats this suggests that a 10 μg dose of GHRP(inip)bbFK-NH₂ would be well above the effective doses-responses range.

There were differences between the anabolic effects of (inip)bbFK-NH₂ in90 vs. 150 day old rats. In the older rats there was no clear effect onliver weight or spleen weight as seen with GHRP (inip)bbFK-NH₂ infusionin the younger rats. (inip)bbFK-NH₂ injections increased thymus weight,suggesting that these GHRPs can stimulate growth of immune tissue andtherefore increase immune function. Other studies have shown that GH andIGF-1 can significantly stimulate immune function, so it would beexpected that GHRP's of this invention, by increasing GH secretion,would also stimulate immune function. Kelley, Ann. N.Y. Acad. Sci.594:95-118 (1990), Clark et al., J. Clin. Invest. 92:540-548 (1993).GHRP (inip)bbFK-NH₂ tended to increase cardiac weight, indicating aneffect on the heart structure and function. GH and IGF-1 have been shownto be efficacious in models of congestive heart failure and this datasuggests that (inip)bbFK-NH₂ would also be effective in the treatment ofcongestive heart failure. Sacca et al., Endocrine Reviews 15:555-573(1994).

The GHRP (inip)bbFK-NH₂ was clearly effective at stimulating an anabolicresponse when delivered by both injections and continuous infusion. Thedelivery of (inip)bbFK-NH₂ by twice daily subcutaneous injection appearsto be an effective method of GHRP delivery. Other methods of deliverythat would produce a similar blood profile of GHRP, for example oraldelivery, delivery to the lung, or other delivery systems designed tomaintain an intermittent exposure to GHRP would also be an effectivemeans of inducing GH secretion and thereby the effects of GH.

The normal serum chemistries indicate that the effects of (inip)bbFK-NH₂can occur without perturbing the normal balance of blood metabolites andions. The one possible exception was the tendency for serum triglycerideto be increased by high dose injections, but not by low dose injectionsof the GHRP (inip)bbFK-NH₂. This may indicate that very high doses of(inip)bbFK-NH₂ can impact the ACTH system and induce corticosteroneactivity. However 10 μg injections of (inip)bbFK-NH₂ did not seem toaffect serum lipids, indicating that this dose, while maximallystimulating GH secretion, has a minimal effect on corticosteronesecretion.

E. Combination GHRP and IGF-1 Treatment of Normal Rats

Normal adult female rats were chosen to study the anabolic effect ofGHRP's 6, (inip)bbF-NH₂, (inip)b(nmb)(bam), and L-692,585 when given incombination with IGF-1. Details of the protocols for this study aredescribed in Example 44.

The body weight gain responses to the GH secretagogues given incombination with IGF-1 (FIG. 27) were much greater than those to the GHsecretagogues given by themselves (FIG. 26). In addition, the responsesto the combination of the GH secretagogues and IGF-1 tended to begreater than to IGF-1 alone.

This study shows for the first time that GHRP has significant anabolicactivity when given in combination with chronically administered IGF-1.Furthermore, there is an additional anabolic benefit of administeringthe combination of GH secretagogues and IGF-1.

3. In Vivo Activity in ZDF Rats

A. Combination GHRP and IGF-1 Therapy in Obese Rats

It is known that IGF-1 inhibits GH secretion by a feedback mechanismeither acting indirectly on the hypothalamus or directly on thepituitary. Tannenbaum et al., Science 220:77-79 (1981). It is also knownthat GHRH induced GH secretion is suppressed by IGF-1 administration(Bermann et al., Program and Abstracts 76th annual Meeting US Endocr.Soc, Abstract 565, (1994). It was however unknown if GHRP could induceGH secretion, or produce effects, in combination with IGF-1administration. The protocol for IGF-1 administration in combinationwith GHRP and GH is provided in Example 43. Rat GHRH, which has beenshown to increase body weight in normal female rats was used as apositive control in the experiment.

Body Weight Gain: The body weight gains plotted against time for alltreatment groups over the entire study (i.e. 24 days) are shown in FIG.22. Body weight gains for the first 7 days for the GHRP (inip)bbFK-NH₂and IGF-1 treatment groups are shown in FIG. 23. Both (inip)bbFK-NH₂ andrhIGF-1 induced significant body weight gain compared to the vehicletreated rats, so that by Day 2 (see FIG. 23) of treatment, the weightgains were statistically significantly greater than the excipienttreated rats ((inip)bbFK-NH₂ : 18.3±1.0 g, rhIGF-1:21.5±0.7 g, and obesecontrols: 13.8±0.4 g). Treatment with rhGH had not increased weight gainby this time (13.6±2.5 g). The weight gain in response to the GHRP(inip)bbFK-NH₂ plus rhIGF-1(26.8+0.8 g) was greater (p<0.05) than thatto the combination of rhGH+rhIGF-1 (23.3±1.2 g). At day 7 of treatmentthese differences in weight gain were maintained. By day 24 of treatment(FIG. 22) the weight gain response to (inip)bbFK-NH₂ plus IGF-1(247.4±7.1 g) was similar to that to rhGH+rhIGF-1 (245.2±4.9 g) and muchgreater than that of obese controls (169.0±1.6 g) and significantlygreater (p<0.05) than for rhIGF-1 treatment alone (232±2 g). It wassurprising that GHRP (inip)bbFK-NH₂ could induce a weight gain whengiven in combination with IGF-1, and that this weight gain was equal tothat of a large dose of rhGH and that the effects of the combination ofGHRP and rhIGF-1 gave weight gains greater than or equal to those of thecombination of rhGH and rhIGF-1.

B. Combination GHRP and IGF-1 Treatment of Diabetic Rats

Obese Zucker Diabetic Fatty (ZDF) rats were chosen to study thediabetogenic effect of GHRP when given in combination with IGF-1.Details of the protocols for this study are described in Example 43.

Blood glucose: High concentrations of blood glucose were used to definethe diabetic state of an animal. Rats were started on treatment beforediabetes had developed (there was no difference between lean and obeseblood glucose values at day 0). FIG. 24 shows the changes with time infasting blood glucose for the 6 obese groups and the lean controls. Byday 14 the obese rats were clearly diabetic (obese controls 218±27 mg %,lean controls 140±3 mg %). Treatment with rhGH gave a greater (p<0.05)increase in blood glucose (504±38 mg %) than did the GHRP (inip)bbFK-NH₂(386±63 mg %). In addition the combination of GHRP (inip)bbFK-NH₂ plusIGF-1 resulted in a blood glucose value of 190±27 mg % comparable tothat for rhGH+IGF-1 treatment of 233±49 mg %.

On Day 24 the blood glucose of the obese diabetic rats had risen to morethat twice that of the lean controls (147±4 mg % vs. 30±57 mg %) andvalues were significantly (p<0.05) higher for rhGH treated rats (725±30mg % ) than for GHRP treated rats (542±37 mg %). This difference betweenrhGH and GHRP was also observed in combination treatment with rhIGF-1.GHRP plus rhIGF-1 resulted in a lower blood glucose measurement (301±53mg %) than did rhGH+rhIGF-1 treatment (512±55 mg %). However the glucosevalues in the GHRP+IGF-1 treated group were elevated (p<0.05) comparedto animals receiving rhIGF-1 alone (177±4 mg %).

Serum Insulin: At Day 0 the levels in obese rats were elevated comparedto lean controls, but there were no differences between the levels inthe obese treatment groups. At week 1 IGF-1 treatment significantlyreduced serum insulin (obese control, 21±2 ng/ml; IGF-1 treated, 8±1ng/ml). By itself GHRP (inip)bbFK-NH₂ elevated serum insulin 35±6 ng/mlas did rhGH treatment (48±6 ng/ml). However the combination of GHRP(inip)bbFK-NH₂ plus IGF-1 significantly lowered insulin (to 10±2 ng/ml)compared to the combination of rhGH plus IGF-1 (25±3 ng/ml, p<0.05).Therefore there was evidence that the combination of GHRP (inip)bbFK-NH₂and IGF-1 stimulated insulin secretion to a lesser extent (was lessdiabetogenic) than the combination of GH and IGF-1.

Insulin Sensitivity: At Day 2 the sensitivity of the animals to insulinwas gauged by measuring blood glucose 30 minutes after an insulinchallenge (FIG. 25). Following insulin, absolute blood glucose valueswere again higher in obese diabetic animals (277±42 mg %) than in leananimals (84±8 mg %). However, blood glucose was now reduced in GHRPtreated animals to levels (323±67 mg %) not different from those of theobese controls. In contrast, in rhGH treated rats, glucose remainedsignificantly (p<0.05) elevated (533±55 mg %) compared to obese controlsor GHRP treated rats. A similar difference between rhGH and GHRPtreatment was seen when they were combined with rhIGF-1 treatment. Bloodglucose was significantly (p<0.05) lower in GHRP+rhIGF-1 treated rats(238±47 mg %) than in rhGH+rhIGF-1 treated rats (388±48 mg %).

These experiments compare the anabolic and diabetogenic effects ofadministered GH and GHRP in a rat model of Type II diabetes. Theseexperiments show for the first time that administered GHRP's havesignificant anabolic activity when given in combination with IGF-1. Thisanabolic activity was equivalent to that induced by treatment with GHplus IGF-1. In contrast, administering GH caused significantly greaterinsulin resistance, as measured by serum glucose and insulin, and by aninsulin challenge, than did administered GHRP, even when they were givenin combination with rhIGF-1. This study shows that the diabetogeniceffect of GHRP (inip)bbFK-NH₂ was significantly less than that of rhGH,at doses that produced similar anabolic effects.

F. Administration

The present invention also provides compositions containing an effectiveamount of compounds of the present invention, including the nontoxicaddition salts, amides and esters thereof, which may, alone, serve toprovide the above-recited therapeutic benefits. Such compositions can beprovided together with physiologically tolerable liquid, gel or soliddiluents, adjuvants and excipients.

The compounds and compositions can be administered to mammals includinghumans in a manner similar to other therapeutic agents. The dosage to beadministered will depend on the usual factors including; age, weight,sex, condition of the patient and route of administration. In general,the dosage required for therapeutic efficacy will range from about 0.001to 1000 μg/kg, more usually 0.01 to 2.5 μg/kg of the host body weight.Alternatively, dosages within these ranges can be administered byconstant infusion over an extended period of time until the desiredtherapeutic benefits have been obtained.

Typically, such compositions are prepared as injectable liquid solutionsor suspensions. Compositions may also be emulsified. The activeingredient is often mixed with diluents or excipients which arephysiologically tolerable and compatible with the active ingredient.Suitable diluents and excipients are, for example, water saline,dextrose, glycerol, or the like, and combinations thereof. In addition,if desired the compositions may contain minor amounts of auxiliarysubstances such as wetting or emulsifying agents, stabilizing orpH-buffering agents, and the like. For a more detailed description ofthe foregoing see a standard pharmaceutical text such as Remington'sPharmaceutical Sciences, Mack Publishing Co. Easton, Pa. (1970).

The compositions of this invention are conventionally administeredparenterally by injection, either subcutaneously or intravenously.Additional formulations which are suitable for other modes ofadministration include suppositories, intranasal aerosols, and, in somecases, oral formulations. For suppositories, traditional binders andexcipients may include, for example, polyalkylene glycols ortriglycerides; such suppositories may be formed from mixtures containingthe active ingredient in the range of 0.5% to 10% preferably 1%-2%. Oralformulations include such normally employed excipients as, for example,pharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharin, cellulose, magnesium carbonate, and the like. Thesecompositions take the form of solutions, suspensions, tablets, pills,capsules, sustained-release formulations, or powders, and contain10%-95% if active ingredient, preferably 25%-70%.

The peptidomimetic compounds may be formulated into the compositions asneutral or salt forms. Pharmaceutically acceptable nontoxic saltsinclude the acid addition salts (formed with the free amino groups) andwhich are formed with inorganic acids such as, for example, hydrochloricor phosphoric acids, or organic acids such as acetic, oxalic, tartaric,mandelic, and the like. Salts formed with the free carboxyl groups maybe derived from inorganic bases such as, for example, sodium, potassium,ammonium, calcium, or ferric hydroxides, and such organic bases asisopropyl amine, 2-ethylamino ethanol, histidine, procaine, and thelike.

EXAMPLES General Experimental

The compounds synthesized via the routes shown in Scheme I-VI followedstandard solid-phase methodologies (Barany, G. and Merrifield, R. B.(1980) in "The Peptides",2,1-284. Gross, E. and Meienhofer, J. Eds.Academic Press, New York.).

The chemical name abbreviations for common reagents and unusual aminoacids used in the examples below are defined as follows (for thedefinition of acronyms specific to the Tables of Example 39, see TableI):

αNal L-α-Naphthylalanine or (L-3-(1-naphthyl)-Alanine)

BOC tert-Butyloxycarbonyl

BOPbenzotriazol-1-yloxy-tri-(dimethylamino)-phosphonium-hexafluorophosphate

BOP-Cl bis(2-oxo-3-oxazolidinyl)phosphinic chloride

CBZ Benzyloxycarbonyl

βNal L-β-Naphthylalanine or (L-3-(2-naphthyl)-Alanine)

DβNal D-β-Naphthylalanine or (D-3-(2-naphthyl)-Alanine)

DCM Dichloromethane

DIPC Diisopropylcarbodiimide

DIPEA Diisopropylethylamine

DMF N,N-Dimethylformamide

DMA N,N-Dimethylacetamide

DMSO Dimethylsulfoxide

EDC 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride

FMOC Fluorenyloxymethylcarbonyl

HBTU [2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium]hexafluorophosphate

HOBt Hydroxybenzotriazole

inip Isonipecotic acid (Piperidine-4-carboxylic acid)

NBHA p-methylbenzhydrylamine

MeOH Methanol

MS Mass Spectrometry

N-Me N-Methyl

NMM N-Methyl morpholine

NMP N-Methyl pyrolidinone

TEA triethylamine

TFA Trifluoroactic acid

THF Tetrahydrofuran

Note: Standard three letter codes are used to designate the naturalamino acids with a "D" placed before it signifying the dextrorotatoryenantiomer (i.e. D-Phe is D-Phenylalanine)

The generally preferred solid phase chemistry protocols for thesynthesis of peptidic compounds of this invention using both BOC- andFMOC- alpha-amine protecting group protocols are shown below. In theabsence of a detailed experimental procedure to the contrary, thefollowing chemistry was employed for the synthesis of compounds in theexamples:

Standard BOC Chemistry Cycle

1) 3×1 min. DCM

2) check ninhydrin, recouple if positive

3) 1×1 min. 45% TFA*

4) 1×25 min. 45% TFA

5) 1×30 sec DCM

6) 1×1 min. MeOH

7) 2×1 min. DCM

8) 1×1 min. 10% TEA/DCM

9) 1×8 min. 10% TEA/DCM

10) 3×1 min. DCM

11) add preactivated amino acid and couple for 1 h

12) go to step 1

Notes

a) For peptide amides, use MBHA resin and start the synthesis at step 7.

b) For the coupling of standard amino acids, premix 3 equiv. of aminoacid, BOP, and HOBt in DMA for 10 min., add 3 equiv. of NMM, then addmixture to the peptide resin. Gentle bubbling of nitrogen into theslurry my means of a glass frit is a preferred method of agitationduring the reaction and washing steps.

c) For coupling to N-α-alkyl-amino acids, use 3 equiv. of amino acid andDIPC, overnight. Alternatively, 3 equiv. of amino acid, BOP-Cl, andDIPEA in DCM, overnight, may be used.

To fully deblock and cleave the peptide from the resin, add (per 1 gresin) 10 mL of HF, 1 mL of anisole, and 0.5 mL of ethylmethylsulfideand stir at 0° C. for 1 h (for peptides containing Trp, 0.25 g ofp-cresol must be added). After the HF is removed, the residue istriturated with ether, collected on a glass frit, and washed severaltimes with ether. The crude peptide is extracted off the resin bywashing successively with 10% HOAc/water, HOAc, acetonitrile, 10%HOAc/water, and water. The combined filtrates are frozen andlyophilized. Purification, preferably via reverse phase (C-18) HPLCusing an acetonitrile (0.1% TFA)/water (0.1% TFA) gradient, provides thepure peptide.

Standard FMOC Chemistry Cycle

1) 5×1 min. DMA

2) check ninhydrin, recouple if positive

3) 1×1 min. 20% piperidine/DCM*

4) 1×15 min. 20% piperidine/DCM

5) 5×1 min. DMA

6) 1×1 min. DCM

7) add preactivated amino acid and couple for 30 min. to 1 h

8) go to step 1

Continue from step 3

4a) 1×30 sec DMA

5a) 1×30 sec DCM

6a) 1×30 sec DMA

7a) 1×30 sec DCM

8a) Add preactivated amino acid and couple for 30 min.-1 h

9a) go to step 1 above

Notes

a) For peptide amides, use FMOC-Am-resin (see below for synthesis),pre-swelled with DCM, and start the synthesis at step 3.

b) For coupling of standard amino acids, premix 3 equiv. of the aminoacid and BOP in DMA/DCM (1:1) for 10 min., add 3 equiv. of NMM, and addthe mixture to the peptide-resin slurry in DMA/DCM (1:1), under gentlenitrogen bubbling.

c) For coupling to N-α-alkyl-amino acids, use 3 equiv. of amino acid andDIPC in DCM, overnight. Alternatively, 3 equiv. of amino acid, BOP-Cl,and DIPEA in DCM, overnight, may be used.

d) To fully deblock and cleave the peptide from the resin, add (per 1 gof resin) 10-15 mL of 95% TFA/triethylsilane (v/v) and shake or stir atroom temperature for 1 h. The TFA is removed under vacuum and theresidue is triturated with ether, collected on a glass frit, and washedseveral times with ether. The crude peptide is extracted off the resinby washing successively with 10% HOAc/water, HOAc, acetonitrile, 10%HOAc/water, and water. The combined filtrates are frozen andlyophilized. Purification, preferably via reverse phase (C-18) HPLCusing an acetonitrile (0.1% TFA)/water (0.1% TFA) gradient, provides thepure peptide.

e) FMOC-Am-resin was prepared as follows: 60.5 g (0.47 mmol/g, 28.4mmol, Advanced Chemtech #SA5002) of aminomethylated polystyrene resinwas placed in a sintered glass funnel reaction vessel and swelled withDCM for 20 min while being agitated with nitrogen bubbling. The solventwas removed by applying a vacuum to the bottom of the funnel and 10% TEAin DCM added. After 20 min., the resin was washed with three portions ofDCM and a solution of 23.0 g of p-[(R,S)-α-[1-(9H-Fluoren-9-yl)methoxyformamido]-2,4-dimethoxybenzyl]-PA (42.6mmol, Novachem) in 100 mL of DMA added. 100 mL of a 1 M solution of DIPCin DCM (100 mmol) was added and the thick suspension agitated for 3.5 h.The resin was washed five times with DMA, twice with DCM, and twice withmethanol. Drying in vacuo gave 70.2 g of FMOC-Am-resin at approximately0.40 mmol/g substitution.

EXAMPLE 1 ##STR50## (inip)-DβNal-DβNal-Phe-Lys-amide, TFA Salt Method A

Step A: (N-ε-BOC)Lys-(Am-resin)

FMOC-Am-resin (10 g, 0.50 mmol/g, 5.0 mmol) was deblocked by agitatingwith 20% piperidine in DMA for 15 min followed by successive washes withDMA (5×) and DCM (1×). The resin gave a positive test with ninhydrin. Asolution of 9.37 g (20.0 mmol) of FMOC-(N-ε-BOC)-L-Lysine, 8.85 g (20.0mmol) of BOP, and 3.31 mL (30.0 mmol) of NMM in 30 mL of DMA was addedand the solution agitated for 1 h. The resin was washed (5×) with DMAand shown to give a negative ninhydrin test. The N-α-FMOC protectinggroup was removed with 20% piperidine in DMA for 15 min, followed bysuccessive washings with DMA (5×) and DCM (1×) to give(N-ε-BOC)Lys-(Am-resin).

Step B: Phe-(N-ε-BOC)Lys-(Am-resin)

A solution of FMOC-L-Phenylalanine (7.75 g, 20.0 mmol) and BOP (8.85 g,20.0 mmol) in 50 mL of DMA/DCM (1:1) was preactivated for 10 min andadded to the (N-ε-BOC)Lys-(Am-resin) from Step A, followed by NMM (3.31mL, 30.0 mmol). After 1 h, the resin was washed with DMA (5×, ninhydrinnegative), deblocked with 20% piperidine in DMA for 15 min, and washedagain with DMA (5×) and DCM (1×) to give Phe-(N-ε-BOC)Lys-(Am-resin),displaying a positive ninhydrin test.

Step C: DβNal-Phe-(N-ε-BOC)-Lys-(Am-resin)

FMOC-D-β-naphthylalanine (4.37 g, 10.0 mmol) and 4.42 g (10.0 mmol) ofBOP in 50 mL of DMA/DCM (1:1) was preactivated for 10 min, 1.65 mL (15.0mmol) of NMM was added, and the mixture added to thePhe-(N-ε-BOC)Lys-(Am-resin) from step B. After agitation for 2 h, theresin was washed with DMA (5×, ninhydrin negative), deblocked with 20%piperidine in DMA for 15 min, and washed again with DMA (5×) and DCM(1×), to give DβNal-Phe-(N-ε-BOC)Lys-Am-resin), displaying a positiveninhydrin test.

Step D: FMOC-DβNal-DβNal-Phe-(N-ε-BOC)Lys-(Am-resin)

FMOC-DβNal (4.37 g, 10.0 mmol) and 4.42 g (10.0 mmol) of BOP in 50 mL ofDMA/DCM (1:1) was preactivated for 10 min and added to theDβNal-Phe-(N-ε-BOC)Lys-(Am-resin) from step C, followed by 1.65 mL (15.0mmol) of NMM. After agitation for 3 h, the resin was washed with DMA(5×, ninhydrin negative), DCM (2×), and methanol (2×). The resin wasdried in vacuo to give 15.6 g ofFMOC-DβNal-DβNal-Phe-(N-ε-BOC)Lys-(Am-resin) with a substitution levelof approximately 0.32 mmol/g.

Step E: N-FMOC-Isonipecotic acid

To a solution of 10.0 g (77.4 mmol) of isonipecotic acid (Aldrich) in 1N sodium carbonate/dioxane (1:1) at 0° C., was added 21.1 g (77.4 mmol)of 9-fluorenylmethylsuccinimidyl carbonate, portionwise. After 14 h, thedioxane was removed in vacuo, and the suspension diluted with 1200 mL ofwater. After extraction with 2 portions of ether (discarded), theaqueous solution was cooled in an ice bath and acidified to pH 3 withconcentrated hydrochloric acid. The slurry was extracted twice withethyl acetate and the combined organics washed with water, brine, driedover anhydrous magnesium sulfate, and filtered. The filtrate wasconcentrated to 500 mL, diluted with 700 mL of hexane, and placed in arefrigerator overnight. The product was collected on a filter and driedin vacuo to give 25.8 g (95%) of N-FMOC-isonipecotic acid as a colorlesssolid.

Step F: (inip)-DβNal-DβNal-Phe-Lys-amide, TFA salt

FMOC-DβNal-DβNal-Phe-(N-ε-BOC)-Lys-(Am-resin) (1.0 g, 0.32 mmol) wasswelled with DCM for 15 min, deblocked with 20% piperidine in DMA for 15min, and washed with DMA (5×) and DCM (1×), to giveDβNal-DβNal-Phe-(N-ε-BOC)-Lys-(Am-resin), displaying a positiveninhydrin test. A preactivated solution of N-FMOC-isonipecotic acid (462mg, 1.32 mmol), 583 mg (1.32 mmol) of BOP, and 0.217 mL (1.98 mmol) ofNMM in 10 mL of DMA/DCM (1:1) was added. After agitation for 2 h, theresin was washed with DMA (5×, ninhydrin negative) and deblocked with20% piperidine in DMA for 15 min. The resin was washed again with DMA(5×) and DCM (3×) then dried in vacuo. The dry resin was suspended in 10mL of TFA and 0.50 mL of triethylsilane added. The mixture was agitatedfor 1 h, concentrated in vacuo, and the resin washed 3× with ether. Thecrude peptide was recovered from the resin by washing with 10% aqueousHOAc, followed by acetonitrile. The combined filtrates were lyophilizedto give 140 mg of a solid. A 70 mg aliquot was purified by reverse phaseHPLC (15-20 μ, 300 Å, Vydac C-18, 1×50 cm, gradient: 23-38% acetonitrile(0.1% TFA) in water (0.1% TFA) in 60 min at 9 mL/min,rt=30 min) to give34 mg of (inip)-DβNal-DβNal-Phe-Lys-amide, TFA salt as a colorlesspowder after lyophilization. MS (electrospray, M+H) 798.4.

Method B

Step A: BOC-(2-Cl-CBZ)Lys-(MBHA-resin)

A 20.0 g sample of MBHA-resin (substitution @ 1.04 mmol/g, 20.8 mmol)was washed successively with NMP, DCM (2×), 5% DIPEA/DCM (1×1 min), 5%DIPEA/DCM (1×10 min), and DCM (4×). A solution of 25.8 g (3 eq) ofBOC-L-(N-ε-(2Cl-CBZ))-Lysine in NMP/DCM (1:1) was added, followed by8.40 g (3 eq) of HOBt in NMP and 62.4 mL (3 eq) of a 1.0 M solution ofDIPC in DCM. After agitation for 1 h, the resin was washed with NMP(1×), DCM (2×), and the coupling judged complete by a ninhydrin test.The resin was neutralized with 5% DIPEA/DCM (1×1 min), 5% DIPEA/DCM(1×10 min), DCM (4×), and capped by the addition of 19.7 mL (10 eq) ofacetic anhydride and 14.4 mL (4 eq) of DIPEA in DCM for 10 min. Washingthe resin with DCM (3×) gave BOC-(2-Cl-CBZ)Lys-(MBHA-resin).

Step B: BOC-Phe-(2-Cl-CBZ)Lys-(MBHA-resin)

Note: The following synthesis cycle was used for all subsequentcouplings to this sample of resin:

1) Deprotect with 50% TFA/DCM for 1 min

2) Deprotect with 50% TFA/DCM for 20 min

3) Wash resin 4× with DCM

4) Neutralize with 5% DIPEA/DCM for 1 min

5) Neutralize with 5% DIPEA/DCM for 5-10 min

6) Wash resin 3× with DCM

7) Wash resin 1× with NMP

8) Preactivate the BOC-amino acid (3 eq) with BOP (3 eq) and HOBt (3 eq)in NMP for 10 min, add NMM (4.5 eq) and transfer to vessel with resin.Couple for 1 h.

9) Wash resin 1× with NMP

10) Wash resin 2× with DCM

11) check ninhydrin for completion of coupling

12) Recouple, if neccessary (steps 4-11)

13) If coupling is complete, proceed with steps 1-11 for coupling of thenext residue.

Specifically, BOC-(2-Cl-CBZ)Lys-(MBHA-resin), vide supra, was deblocked,washed, and coupled with 16.4 g (3 eq) of BOC-L-Phe, 27.6 g of BOP, 8.4g of HOBt, and 10.3 mL of NMM for 40 min to giveBOC-Phe-(2-Cl-CBZ)Lys-(MBHA-resin) (ninhydrin negative).

Step C: BOC-DβNal-Phe-(2-Cl-CBZ)Lys-(MBHA-resin)

The above sample of BOC-Phe-(2-Cl-CBZ)Lys-(MBHA-resin) was deblocked,washed, and coupled with 13.1 g (2 eq) of BOC-D-β-naphthylalanine, 18.3g of BOP, 5.6 g of HOBt, and 6.85 mL of NMM for 1 h giving an incompletereaction (ninhydrin positive). The resin was recoupled (c.f. step 12above) using 6.55 g (1 eq) of BOC-D-β-napthylalanine, 9.2 g of BOP, 2.8g of HOBt, and 3.43 mL of NMM for 1 h to giveBOC-DβNal-Phe-(2-Cl-CBZ)Lys-(MBHA-resin) (ninhydrin negative).

Step D: BOC-DβNal-DβNal-Phe-(2-Cl-CBZ)Lys-(MBHA-resin)

The above sample of BOC-DβNal-Phe-(2Cl-CBZ)Lys-MBHA-resin) wasdeblocked, washed, and coupled with 13.1 g (2 eq) ofBOC-D-β-naphthylalanine, 18.3 g of BOP, 5.6 g of HOBt, and 6.85 mL ofNMM for 3 h giving BOC-DβNal-DβNal-Phe-(2-Cl-CBZ)Lys-(MBHA-resin)(ninhydrin negative).

Step E: N-BOC-Isonipecotic acid

To a cold solution of 12.4 g (0.31 mmol, 1.0 eq) of sodium hydroxide in300 ml of water and 600 ml of dioxane was added 40.0 g (0.31 mmol, 1.0eq) of isonipecotic acid, followed by 84.0 g (38 mmol, 1.2 eq) ofdi-t-butyl dicarbonate. The mixture was stirred at ambient temperaturefor 5 h then partitioned between ethyl acetate and 0.5 N citric acid.The organic phase was washed with water, brine, dried over sodiumsulfate, and concentrated. The crystalline product was collected byfiltration, washed with hexane, and dried under vacuum. Yield: 64.0 g(90%), 1H NMR (300 MHz, CDCl₃) δ 10.78 (1H, exc), 4.0 (2H, d), 2.85(2H,t), 2.48 (1H, m), 1.9 (2H, m), 1.65 (2H, m), 1.42 (9H, s). MS (FAB,M+H) 230.1.

Step F: BOC-(inip)-DβNal-DβNal-Phe-(2-Cl-CBZ)Lys-MBHA-resin)

The above sample of BOC-DβNal-DβNal-Phe-(2Cl-CBZ)Lys-(MBHA-resin) wasdeblocked, washed, and coupled with 9.54 g (2 eq) of BOC-isonipecoticacid, 18.3 g of BOP, 5.6 g of HOBt, and 6.85 mL of NMM for 1 h, givingBOC-(inip)-DβNal-DβNal-Phe-(2-Cl-CBZ)Lys-(MBHA-resin) (ninhydrinnegative).

Step G: (inip)-DβNal-DβNal-Phe-Lys-amide

The above sample ofBOC-(inip)-DβNal-DβNal-Phe-(2-Cl(CBZ)Lys-(MBHA-resin) was deblocked,washed, neutralized, washed with DCM (4×), ethanol (4×), and dried invacuo to yield 40.4 g of(inip)-DβNal-DβNal-Phe-(2Cl-CBZ)Lys-(MBHA-resin). The resin wastransfered to a teflon reaction vessel and stirred with a mixture of 200mL of HF, 20 mL of anisole, and 20 mL of ethyl methyl sulfide at 0° C.for 1 h. The volitiles were removed in vacuo, the resin transfered to aglass fritted funnel and washed repeatedly with ether. The product wasextracted from the resin by successive washings with 10% HOAc/water,acetonitrile, and water. The washings were pooled and lyophilized toafford 7.5 g of a powder that was purified by reverse phase HPLC (15-20μ, 300 Å, Vydac C-18, 5×25 cm, gradient: 25-45% acetonitrile (0.1% TFA)in water (0.1% TFA) in 200 min at 50 mL/min, rt-=50 min) to give 4.39 gof (inip)-DβNal-DβNal-Phe-Lys-amide, TFA salt as a colorless powderafter lyophilization. MS (electrospray, M+H) 798.7.

EXAMPLE 2 ##STR51## (4-aminobutanoyl)-DβNal-DβNal-Phe-Lys-amide, TFASalt

FMOC-DβNal-DβNal-Phe-(N-ε-BOC)Lys-(Am-resin) (1.0 g, 0.32 mmol), fromExample 1, Method A, step D, was swelled with DCM for 15 min, deblockedwith 20% piperidine in DMA for 15 min, and washed with DMA (5×) and DCM(1×) to give DβNal-DβNal-Phe-(N-ε-BOC)Lys-(Am-resin), displaying apositive ninhydrin test. A preactivated solution of N-BOC-4-aminobutyricacid (403 mg, 1.98 mmol), 875 mg (1.98 mmol) of BOP, and 0.33 mL (2.97mmol) of NMM in 15 mL of DMA/DCM (1:1) was added. After agitation for 1h, the resin was washed with DMA (5×, ninhydrin negative), DCM (3×),MeOH (2×) and dried in vacuo. The dry resin was suspended in 10 mL ofTFA and 0.50 mL of triethylsilane added. The mixture was agitated for 1h, concentrated in vacuo, and the resin washed with ether. The crudepeptide was recovered from the resin by washing with 10% aq HOAc,followed by acetonitrile. The combined filtrates were lyophilized togive 245 mg of a solid. A 50 mg aliquot was purified by reverse phaseHPLC (15-20 μ, 300 Å, Vydac C-18, 1×50 cm, gradient: 23-38% acetonitrile(0.1% TFA) in water (0.1% TFA) in 60 min at 9 mL/min, rt=25 min) to give28 mg of (4-aminobutanoyl)-DβNal-DβNal-Phe-Lys-amide, TFA salt as acolorless powder after lyophilization. MS (electrospray, m+h) 772.4.

EXAMPLE 3 ##STR52##[4-(N-Methylamino)butanoyl]-DβNal-DβNal-Phe-Lys-amide, TFA Salt

Step A: BOC-4-(N-Methylamino)butyric Acid

To a 0° C. solution of 5.00 g (24.6 mmol) of BOC-4-aminobutyric acid in75 mL of dry THF, was added 12.2 mL (197 mmol) of methyl iodide followedby 2.95 g (73.8 mmol, 60% dispersion in mineral oil) of sodium hydride,portionwise. The reaction was rapidly stirred at room temperature for 12h and quenched by the careful addition of water. The mixture waspartitioned between ether and water, and the organic phase extractedwith 1 N aq sodium bicarbonate. The combined aqueous phases were chilledand acidified to pH 3 with 1 N sodium hydrogen sulfate, then extractedwith two portions of ethyl acetate. The combined organics were washedsuccessively with water, 5% aq sodium thiosulfate, water, brine, andthen dried over anhydrous magnesium sulfate. Concentration in vacuoafforded 5.00 g (94%) of BOC-4-(N-methylamino)butyric acid. ¹ H NMR:(300 MHz, CDCl₃) δ 3.28 (2H, bt, J=7 Hz), 2.84 (3H,s), 2.36 (2H,t,J=7.5Hz), 1.85 (2H, m), 1.45 (9H, s).

Step B: [4-(N-Methylamino)butanoyl]-DβNal-DβNal-Phe-Lys-amide, TFA salt

Following the procedure of Example 2,FMOC-DβNal-DβNal-Phe-(N-ε-BOC)Lys-(Am-resin) (0.50 g, 0.17 mmol, (fromExample 1, Method A, Step D) was deblocked and coupled toBOC-4-(N-methylamino)butyric acid (Step A) (147 mg, 0.68 mmol) using 300mg (0.68 mmol) of BOP and 0.11 mL (1.02 mmol) of NMM. Cleavage from theresin afforded 120 mg of a solid after lyophilization. A 62 mg aliquotwas purified by reverse phase HPLC (15-20 μ, 300 Å, Vydac C-18, 1×50 cm,gradient: 23-38% acetonitrile (0.1% TFA) in water (0.1% TFA) in 60 minat 9 mL/min, rt=23 min) to give 27 mg of pure[4-(N-methylamino)butanoyl]-DβNal-DβNal-Phe-Lys-amide, TFA salt. MS(electrospray, M+H) 786.4.

EXAMPLE 4 ##STR53## (inip)-(N-Me-DβNal)-DTrp-Phe-Lys-amide, TFA Salt

Step A: DTrp-Phe-(N-ε-BOC)Lys-(Am-resin)

A 1 g (approx. 0.4 mmol) sample of Phe-(N-ε-BOC)Lys-(Am-resin) (Example1, Method A, Step B) was reacted with a preactivated solution of 0.85 g(2.0 mmol) of FMOC-D-Tryptophan, 0.88 g of BOP, and 0.33 mL of NMM for 1h. Washing and deprotection as per the general protocol above gaveDTrp-Phe-(N-ε-BOC)Lys-(Am-resin), displaying a positive ninhydrin test.

Step B: BOC-N-Methyl-D-β-naphthylalanine (BOC-N-Me-DβNal)

To a cold (0° C.), stirred, THF solution of N-BOC-D-β-naphthylalanine(15.0 g, 47.6 mmol) and methyl iodide (14.8 ml, 238 mmol) was addedsodium hydride (5.70 g of 60% dispersion in mineral oil, 143 mmol) inportions over 45 min. The mixture was allowed to warm slowly to ambienttemperature over 16 h then partially concentrated and poured into 1 L ofdilute aqueous sodium bicarbonate. Neutral species were extracted intoethyl acetate and discarded. The aqueous phase was acidified with citricacid and the separated product extracted into ethyl acetate, washed withdilute sodium bisulfite, brine, dried over magnesium sulfate, andconcentrated. The crude product was crystallized from DCM/hexane toyield 14.9 g (95%) of the title compound. ¹ H NMR (300 MHz, CDCl₃,rotational isomers evident) δ 1.31 (9H, 2s, BOC), 2.70 (3H, 2s, N-Me),3.32 (2H, m, CH₂ Ar), 4.8 (1H, m, CH), 7.54 (7H, m, Ar). MS (FAB,M+H)330.2.

Step C: FMOC-N-Methyl-D-β-naphthylalanine (FMOC-N-Me-DβNal)

BOC-N-methyl-D-βnaphthylalanine (10.0 g, 30.4 mmol) was dissolved in TFA(100 ml) and stirred for 1 h. The TFA was removed under vacuum and theresidue combined with 9-fluorenylmethyl-succinimidylcarbonate (13.3 g,40.0 mmol), potassium carbonate (6.2 g, 45 mmol), THF (300 ml), water(124 ml), and stirred at ambient temperature for 18 h. The reactionmixture was partitioned between ethyl acetate and dilute aq HCl, and theorganic phase washed with brine, dried over magnesium sulfate, andconcentrated. The crystalline product was collected by filtration andwashed with hexane to yield 12.9 g (94%) of the title compound. ¹ H NMR(300 MHz, DMSO-d₆, rotational isomers evident) δ 2.70 (3H, 2s, NMe),3.20 (2H, m, CH₂ Ar), 4.18 (3H, m, OCH₂ and CHAr), 4.90 (1H, m, COCHN),7.0 to 8.0 (15H, m, Ar), 13.0 (1H, s, COOH). .sub.[α]²⁰ _(d) =+48.0°(c=1.625 in MeOH/DCM 1:1). MS (FAB, M+H) 452.3.

Step D: (N-Me-DβNal)-DTrp-Phe-(N-ε-BOC)Lys-(Am-resin)

The DTrp-Phe-(N-ε-BOC)Lys-(Am-resin) from Step A was agitated with apreactivated solution of 0.68 g (1.5 mmol) of FMOC-(N-Me-DβNal), 0.88 gof BOP, and 0.33 mL of NMM for 3 h. Washing and deprotection as per thegeneral FMOC protocol above gave(N-Me-DβNal)-DTrp-Phe-(N-ε-BOC)Lys-(Am-resin) displaying a faint orangeninhydrin test.

Step E: (inip)-(N-Me-DβNal)-DTrp-Phe-Lys-amide, TFA salt

(N-Me-DβNal)-DTrp-Phe-(N-ε-BOC)Lys-(Am-resin) (1.0 g, 0.32 mmol, Step D)was treated with a preactivated solution of 0.88 g (2.5 mmol) ofN-FMOC-isonipecotic acid, 1.10 g of BOP, and 0.66 mL of NMM for 4 h.After washing with DMA (5×), a ninhydrin test showed the reaction to beincomplete. The resin was recoupled using 0.88 g (2.5 mmol) ofN-FMOC-isonipecotic acid, 0.56 g of BOP-Cl, and 0.87 mL of DIPEA for 12h (ninhydrin negative). The resin was washed, deblocked with 20%piperidine in DMA, washed, and dried in vacuo. Cleavage with TFA andextraction as per the general protocol gave 75 mg of a solid afterlyophilization, which was purified by reverse phase HPLC (15-20 μ, 300Å, Vydac C-18, 1×50 cm, gradient: 17-32% acetonitrile (0.1% TFA) inwater (0.1% TFA) in 60 min at 9 mL/min, rt=45 min) to give 23 mg ofimpure product. Rechromatography (15-20 μ, 300 Å, Vydac C-18, 1×50 cm,gradient: 35-55% methanol (0.1% TFA) in water (0.1% TFA) in 80 min at 10mL/min, rt=30 min) gave 11 mg of the pure title compound. MS(electrospray,M+H) 801.6.

EXAMPLE 5 ##STR54## (inip)-(N-Me-DβNal)-DβNal-Phe-Lys-amide, TFA salt

Step A: (N-Me-DβNal)-DβNal-Phe-(2-Cl-CBZ)Lys-(MBHA-resin)

A 0.8 g (approx. 0.3 mmol) sample ofBOC-DβNal-Phe-(2-Cl-CBZ)Lys-(MBHA-resin) (Example 1, Method B, Step C)was deblocked, washed, and reacted with a preactivated DMA solution of0.33 g (1.0 mmol) of BOC-(N-Me-DβNal) (Example 4, step B), 0.44 g ofBOP, 0.14 g of HOBt, and 0.11 mL of NMM for 1.5 h. Washing anddeprotection as per the general BOC protocol above gave the titlecompound displaying a light orange, positive ninhydrin test.

Step B: (inip)-(N-Me-DβNal)-DβNal-Phe-Lys-amide, TFA salt

The intermediate from step A was treated with 0.23 g (1.0 mmol) ofN-BOC-isonipecotic acid (Example 1, Method B, Step E), 0.29 g of BOP-Cl,and 0.20 mL of DIPEA in DCM for 12 h. Washing, drying, and cleavage asper the general BOC protocol above gave 250 mg of a powder. A 102 mgaliquot was purified by reverse phase HPLC (15-20 μ, 300 Å, Vydac C-18,1×50 cm, gradient: 23-38% acetonitrile (0.1% TFA) in water (0.1% TFA) in60 min at 9 mL/min, rt=32 min) to give 23 mg of the title compound. MS(electrospray,M+H) 812.4.

EXAMPLE 6 ##STR55## (inip)-DβNal-(N-Me-DβNal)-Phe-Lys-amide, TFA salt

Step A: (N-Me-DβNal)-Phe-(2-Cl-CBZ)Lys-(MBHA-resin)

A 0.80 g (approx. 0.30 mmol) sample of BOC-Phe-(2-Cl-CBZ)Lys-(MBHA-resin(Example 1, Method B, Step B) was deblocked, washed, and reacted with apreactivated DMA solution of 0.33 g (1.0 mmol) of BOC-(N-Me-DβNal)(Example 4, step B), 0.44 g of BOP, 0.14 g of HOBt, and 0.11 mL of NMMfor 1.5 h. Washing and deprotection as per the general BOC protocolabove gave the title compound displaying a light orange positiveninhydrin test.

Step B: DβNal-(N-Me-DβNal)-Phe-(2-Cl-CBZ)Lys-(MBHA-resin)

The intermediate from step A was treated with 0.32 g (1.0 mmol) ofN-BOC-D-β-naphthylalanine, 0.29 g of BOP-Cl, and 0.20 mL of DIPEA in DCMfor 12 h. Washing and deprotection as per the general BOC protocol abovegave the title compound displaying a positive ninhydrin test.

Step C: (inip) -DβNal-(N-Me-DβNal)-Phe-Lys-amide, TFA salt

The intermediate from step B was reacted with a preactivated DMAsolution of 0.23 g (1.0 mmol) of N-BOC-isonipecotic acid, 0.44 g of BOP,0.14 g of HOBt, and 0.11 mL of NMM for 1 h. Deblocking, washing, drying,and HF cleavage, as per the general BOC protocol above gave 200 mg of apowder. A 67 mg aliquot was purified by reverse phase HPLC (15-20 μ, 300Å, Vydac C-18, 1×50 cm, gradient: 13-28% acetonitrile (0.1% TFA) inwater (0.1% TFA) in 60 min at 9 mL/min, rt=30 min) to give 25 mg of thetitle compound. MS (electrospray,M+H) 812.4.

EXAMPLE 7 ##STR56## (5-Aminovaleryl)-(N-Me-DβNal)-DβNal-Phe-Lys-amide,TFA salt

Step A: BOC-(N-Me-D-DβNal)-DβNal-Phe-(2-Cl-CBZ)Lys-(MBHA-resin)

A 0.33 mmol sample of BOC-DβNal-Phe-(2-Cl-CBZ)Lys-(MBHA-resin) (Example1, Method B, Step C) was treated with TFA deblock, neutralized, washed,and coupled with 3 eq of BOC-(N-Me-DβNal) (from Example 4, Step B), 3 eqof BOP, 3 eq of HOBt and 4.5 eq of NMM in DMA/DCM for 1 h. The resin waswashed with DMA (5×) to give the title compound (ninhydrin negative).

Step B: (5-Aminovaleryl)-(N-Me-DβNal)-DβNal-Phe-Lys-amide, TFA salt

The above sample of BOC-(N-Me-DαNal)-DβNal-Phe-(2Cl-CBZ)Lys-(MBHA-resin)was deblocked with TFA, neutralized, washed, and coupled with 4 eq ofN-BOC-5-aminovaleric acid, 4 eq of BOP-Cl, and 6 eq of DIPEA in DCMovernight. After confirming complete coupling with the ninhydrin test,the peptide was deblocked with TFA, washed, and dried in vacuo to give(5-aminovaleryl)-(N-Me-DβNal)-DβNal-Phe-(2-Cl-CBZ)Lys-(MBHA-resin). Thepeptide was cleaved from the resin and lyophilized, using the methodsdescribed in the general procedure, to provide 193 mg of a crude solid.A 53 mg aliquot was purified by reverse phase HPLC (15-20 μ, 300 Å,Vydac C-18, 1×50 cm, gradient: 23-38% acetonitrile (0.1% TFA) in water(0.1% TFA) in 60 min at 9 mL/min, rt=43 min) to give 7.4 mg of pure(5-aminovaleryl)-(N-Me-DβNal)-DβNal-Phe-Lys-amide, TFA salt as acolorless powder after lyophilization. MS (electrospray, M+H) 800.2.

EXAMPLE 8 ##STR57## (inip)-DβNal-DβNal-αNal-Lys-amide, TFA salt

Step A: BOC-αNal-(2-Cl-CBZ)Lys-(MBHA-resin)

A 1.0 g sample of BOC-(2-Cl-CBZ)Lys-(MBHA-resin) from Example 1, MethodB, Step A, was deblocked, washed, neutralized with 5% DIPEA/DCM, washed,and coupled with 945 mg (3 mmol) of BOC-L-α-napthylalanine, 1.32 g ofBOP, and 0.45 mL of NMM for 1 h, givingBOC-αNal-(2Cl-CBZ)Lys-(MBHA-resin) (ninhydrin negative).

Step B: BOC-DβNal-αNal-(2-Cl-CBZ)lys-(MBHA-resin)

The intermediate from Step A was deblocked, washed, neutralized with 5%DIPEA/DCM, washed, and coupled with 945 mg (3 mmol) ofBOC-D-β-napthylalanine, 1.32 g of BOP, and 0.45 mL of NMM for 1 h,giving BOC-DβNal-αNal-(2-Cl-CBZ)Lys-(MBHA-resin) (ninhydrin negative).

Step C: BOC-DβNal-DβNal-αNal-(2-Cl-CBZ)Lys-(MBHA-resin)

The resin from Step B was deblocked, washed, neutralized with 5%DIPEA/DCM, washed, and coupled with 945 mg (3 mmol) ofBOC-D-β-napthylalanine, 1.32 g of BOP, and 0.45 mL of NMM for 1 h,giving BOC-DβNal-DβNalαNal-(2-Cl-CBZ)Lys-(MBHA-resin) (ninhydrinnegative).

Step D: (inip)-DβNal-DβNal-αNal-(2Cl-CBZ)Lys-(MBHA-resin)

The resin from Step C was deblocked, washed, neutralized with 5%DIPEA/DCM, washed, and coupled with 690 mg (3 mmol) ofN-BOC-isonipecotic acid, 1.32 g of BOP, and 0.45 mL of NMM for 1 h,giving BOC-(inip)-DβNal-DβNal-αNal-(2-Cl-CBZ)Lys-(MBHA-resin) (ninhydrinnegative). The resin was deblocked, washed with methanol, and dried invacuo to give 1.1 g of the title compound.

Step E: (inip)-DβNal-DβNal-αNal-Lys-amide, TFA salt

The above resin (1.1 g) was cleaved with HF according to the generalprocedure to afford 87 mg of a solid that was purified by reverse phaseHPLC (15-20 μ, 300 Å, Vydac C-18, 1×50 cm, gradient: 25%-40%acetonitrile (0.1% TFA) in water (0.1% TFA) in 60 min at 9 mL/min,rt=min) to give 11 mg of pure (inip)-DβNal-DβNal-αNal-Lys-amide, TFAsalt. MS (electrospray,M+H) 848.4.

EXAMPLE 9 ##STR58##(4-Aminobutanoyl)-DβNal-DβNal-DβNal-[N-(3-aminopropyl)] amide, TFA salt

Step A: 1,3-Propanediamine-carbonylbenzyloxy-resin (PDA-COO-resin)

Hydroxymethyl resin (10 g, 1 meq/g, 100-14 200 mesh, Bachem RMIS35) waswashed with 5% DIPEA/DCM, toluen (6×), suspended in 60 ml of toluene andgently agitated with nitrogen bubbling. A solution of phosgene intoluene (70 mL, 1.93 m, Fluka) was added and, after 20 min, the resinwas washed with toluene (6×), resuspended in 60 mL of dry THF, and1,3-Propanediamine (5.0 g, Fluka) was added. The mixture was agitatedfor 1 h, washed with DMA (5×), and shown to give a positive ninhydrintest. A solution of 10% TFA in DCM was added and the resin was washedwith DCM (4×), methanol (2×), and dried in vacuo.

Step B: BOC-βNal-(PDA-COO-resin)

A 3.0 g sample of PDA-COO-resin was neutralized with 5% DIPEA/DCM,washed, and coupled with 2.84 g (9.0 mmol) of BOC-βNal, 3.96 g of BOP,and 1.5 mL of NMM for 1 h (ninhydrin negative). After washing with DCM,the resin was washed with methanol and dried.

Step C: BOC-DβNal-βNal-(PDA-COO-resin)

A 1.0 g sample of the resin from Step B was deblocked, washed,neutralized with 5% DIPEA/DCM, washed, and coupled with 945 mg (3 mmol)of BOC-DβNal, 1.32 g of BOP, and 0.45 mL of NMM for 1 h, givingBOC-DβNal-βNal-(PDA-COO-resin) (ninhydrin negative).

Step D: BOC-DβNal-DβNal-βNal-(PDA-COO-resin)

The above sample was washed, deblocked, washed, neutralized with 5%DIPEA/DCM, washed, and coupled with 945 mg (3 mmol) of BOC-DβNal, 1.32 gof BOP, and 0.45 mL of NMM for 1 h givingBOC-DβNal-DβNal-βNal-(PDA-COO-resin) (ninhydrin negative).

Step E: (4-Aminobutanoyl)-DβNal-DβNal-βNal-(PDA-COO-resin)

The above sample was washed, deblocked, washed, neutralized with 5%DIPEA/DCM, washed and coupled with 609 mg (3 mmol) ofN-BOC-4-aminobutyric acid, 1.32 g of BOP, and 0.45 mL of NMM for 1 h(ninhydrin negative). The resin was washed, deblocked, washed withmethanol, and dried in vacuo to give 1.3 g of the title compound.

Step F: (4-Aminobutanoyl)-DβNal-DβNal-βNal-[N-(3-aminopropyl)] amide,TFA salt

The above resin (1.3 g) was cleaved with HF to afford 225 mg of a solidafter lyophilization. A 60 mg sample was purified by reverse phase HPLC(15-20 μ, 300 Å, Vydac C-18, 1×50 cm, gradient: 25-40% acetonitrile(0.1% TFA) in water (0.1% TFA) in 60 min at 9 mL/min, rt=30 min) to give29 mg of the title compound. MS (electrospray,M+H) 751.4.

EXAMPLE 10 ##STR59## DβNal-βNal-βNal-[N-(3-aminopropyl)] amide, TFA salt

Step A: BOC-βNal-βNal-(PDA-COO-resin)

A 2 g sample of BOC-βNal-(PDA-COO-resin) (from Example 9, Step B) waswashed, deblocked, washed, neutralized with 5% DIPEA/DCM, washed, andcoupled with 1.89 g (6.0 mmol) of BOC-βNal, 2.64 g of BOP, and 0.90 mLof NMM for 1 h, giving BOC-βNal-βNal-(PDA-COO-resin) (ninhydrinnegative). The resin was washed with methanol, and dried in vacuo togive the title compound.

Step B: BOC-DβNal-βNal-βNal-(PDA-COO-resin)

One gram of the above sample was deblocked, washed, neutralized with 5%DIPEA/DCM, washed, and coupled with 945 mg (3 mmol) of BOC-DβNal, 1.32 gof BOP, and 0.45 mL of NMM for 1 h, givingBOC-DβNal-βNal-βNal-(PDA-COO-resin) (ninhydrin negative). The resin waswashed, deblocked, washed with methanol, and dried in vacuo.

Step C: DβNal-βNal-βNal-[N-(3-aminopropyl)] amide, TFA salt

The above resin (1.15 g) was cleaved with HF to afford 99 mg of a solidafter lyophilization. The sample was purified as per Example 2 (gradient27%-42% in 60 min (rt=22 min)) to give 58 mg ofDβNal-βNal-βNal-[N-(3-aminopropyl)] amide, TFA salt. MS(electrospray,M+H) 666.4.

EXAMPLE 11 ##STR60## Acetyl-DβNal-βNal-βNal-[N-(3-aminopropyl)] amide,TFA salt

Step A: Acetyl-DβNal-βNal-βNal-(PDA-COO-resin)

A 1.0 g sample of the product of Example 10, Step B was deblocked,neutralized with 5% DIPEA/DCM, and coupled with acetic anhydride (2 mL)in 5% DIPEA/DCM (10 mL) giving Acetyl-DβNal-βNal-βNal-(PDA-COO-resin)(ninhydrin negative). The resin was washed with methanol and dried invacuo to give 1.23 g of resin.

Step B: Acetyl-DβNal-βNal-βNal-[N-(3-aminopropyl)] amide, TFA salt

The above resin (1.23 g) was cleaved with HF to afford 155 mg of apowder after lyophilization. A 68 mg sample was purified as per Example2 (gradient 30%-45% in 60 min (rt=40 min)) to give 31 mg of the titlecompound. MS (electrospray,M+H) 708.4.

EXAMPLE 12 ##STR61## (5-Aminovaleryl)-DPhe-βNal-βNal-Lys-amide, TFA salt

Step A: βNal-(N-ε-BOC)Lys-(Am-resin)

A 0.5 g (approx. 0.25 mmol) sample of (N-ε-BOC)Lys-(Am-resin) (Example1, Method A, Step A) was reacted with a preactivated solution of 0.44 g(1.0 mmol) of FMOC-L-βNal, 0.44 g of BOP, and 0.17 mL of NMM for 1 h.Washing and deprotection as per the general protocol above gave thetitle compound.

Step B: βNal-βNal-(N-ε-BOC)Lys-(Am-resin)

The product of step A was reacted with a preactivated solution of 0.44 g(1.0 mmol) of FMOC-L-βNal, 0.44 g of BOP, and 0.17 mL of NMM for 1 h.Washing and deprotection as per the general protocol above gave thetitle compound.

Step C: DPhe-βNal-βNal-(N-ε-BOC)Lys-(Am-resin)

The produce of step B was reacted with a preactivated solution of 0.39 g(1.0 mmol) of FMOC-D-Phenylalanine, 0.44 g of BOP, and 0.17 mL of NMMfor 1 h. Washing and deprotection as per the general protocol above gavethe title compound.

Step D: (5-Aminovaleryl)-DPhe-βNal-βNal-Lys-amide, TFA salt

The product of step C was reacted with a preactivated solution of 0.22 g(1.0 mmol) of N-BOC-5-aminovaleric acid, 0.44 g of BOP, and 0.17 mL ofNMM for 1 h. Washing, drying, and cleavage as per the general FMOCprotocol above gave 100 mg of a powder. A 51 mg aliquot was purified byreverse phase HPLC (15-20 μ, 300 Å, Vydac C-18, 1×50 cm, gradient:25-40% acetonitrile (0.1% TFA) in water (0.1% TFA) in 60 min at 9mL/min, rt=25 min) to give 23 mg of the title compound. MS(electrospray, M+H) 786.5.

EXAMPLE 13 ##STR62##(5-Aminovaleryl)-DPhe-βNal-βNal-[N-(4-aminobutyl)]amide, TFA salt

Step A: 1,4 Butanediamine-carbonylbenzyloxy-resin (BDA-COO-resin

Hydroxymethyl resin (10 g, 1 meq/g, 100-200 mesh, Bachem RMIS35) waswashed with 5% DIPEA/CM, toluene (6×), suspended in 60 ml of toluene andgently agitated with nitrogen bubbling. A solution of phosgene intoluene (70 mL, 1.93 M, Fluka) was added and, after 20 min, the resinwas washed with toluene (6×), resuspended in 60 mL of dry THF, and1,4-Diaminobutane (5.0 g, Fluke) was added. The mixture was agitated for1 h, washed with DMA (5×), and shown to give a positive ninhydrin test.A solution of 10% TFA in DCM was added and the resin was washed with DCM(4×), methanol (2×), and dried in vacuo.

Step B: BOC-βNal-(BDA-COO-resin)

A 1 g sample of BDA-COO-resin from Step A was neutralized with 5%DIPEA/DCM, washed, and coupled with 945 mg (3 mmol) of BOC-βNal, 1.32 gof BOP, and 0.45 mL of NMM for 1 h, giving BOC-βNal-(BDA-COO-resin)(ninhydrin negative).

Step C: BOC-βNal-βNal-(BDA-COO-resin)

The above sample was deblocked, washed, neutralized with 5% DIPEA/DCM,washed, and coupled with 945 mg (3 mmol) of BOC-βNal, 1.32 g of BOP, and0.45 mL of NMM for 1 h giving BOC-βNal-βNal-(BDA-COO-resin) (ninhydrinnegative).

Step D: BOC-DPhe-βNal-βNal-(BDA-COO-resin)

The above sample was deblocked, washed, neutralized with 5% DIPEA/DCM,washed, and coupled with 795 mg (3 mmol) of BOC-D-Phenylalanine, 1.32 gof BOP, and 0.45 mL of NMM for 1 h, givingBOC-DPhe-βNal-βNal-(BDA-COO-resin) (ninhydrin negative).

Step E: BOC-(5Aminovaleryl)-DPhe-βNal-βNal-(BDA-COO-resin)

The the resin from Step D was deblocked, washed, neutralized with 5%DIPEA/DCM, washed, and coupled with 621 mg (3 mmol) ofN-BOC-5-aminovaleric acid, 1.32 g of BOP, and 0.45 mL of NMM for 1 hgiving BOC-(5-aminovaleryl)-DPhe-βNal-βNal-(BDA-COO-resin) (ninhydrinnegative). The resin was washed, deblocked, washed with methanol, anddried in vacuo.

Step F: (5-aminovaleryl)-DPhe-βNal-βNal-[N-(4-aminobutyl)] amide, TFAsalt

The above resin (1.1 g) was deblocked with HF to afford 72 mg of solidafter lyophilization. The sample was purified as per Example 2 with a25%-40% gradient (r=22 min) to give 16 mg of the title compound. MS(electrospray,M+H) 729.5.

EXAMPLE 14 ##STR63## (inip)-DβNal-DβNal-Phe-amide, TFA salt

Step A: Phe-(MBHA-resin)

An 8.0 g sample of MBHA resin (substitution @0.57 mmol/g, 4.56 mmol) wasneutralized, washed, and reacted with a preactivated DMA solution of5.30 g (13.7 mmol) of BOC-L-Phenylalanine, 6.05 g of BOP, 1.85 g ofHOBt, and 1.50 mL of NMM for 1.5 h. Washing and deprotection as per thegeneral BOC protocol above gave the title compound.

Step B: DβNal-Phe-(MBHA-resin)

The intermediate from step A was reacted with a preactivated DMAsolution of 2.87 g (9.12 mmol) of N-BOC-D-βnaphthylalanine, 4.03 g ofBOP, 1.23 g of HOBt, and 1.00 mL of NMM for 1.5 h. Washing anddeprotection gave the title compound.

Step C: DβNal-DβNal-Phe-(MBHA-resin)

The intermediate from step B was reacted with a preactivated DMAsolution of 2.87 g (9.12 mmol) of N-BOC-D-β-naphthylalanine, 4.03 g ofBOP, 1.23 g of HOBt, and 1.00 mL of NMM for 1.5 h. Washing anddeprotection as per the general BOC protocol above gave the titlecompound.

Step D: (inip)-DβNal-DβNal-Phe0amide, TFA salt

One half of the intermediate resin from step C (2.28 mmol) was reactedwith a preactivated DMA solution of 1.57 g (6.84 mmol) ofN-BOC-isonipecotic acid, 3.02 g of BOP, and 1.00 mL of NMM for 3 h.Deblocking, washing, drying, and HF cleavage as per the general BOCprotocol above gave 560 mg of a powder which was purified by reversephase HPLC (15-20 μ, 300 Å, Vydac C-18, 1×50 cm, gradient: 30-44%acetonitrile (0.1% TFA) in water (0.1% TFA) in 150 min at 9 mL/min,rt=60 min) to give 436 mg of the title compound. MS (electrospray,M+H)670.4.

EXAMPLE 15 ##STR64## (inip)-DβNal-DTrp-Phe-amide, TFA salt

Step A: DTrp-Phe-(MBHA-resin)

A 2.0 g (approx. 1.14 mmol) sample of Phe-(MBHA-resin) (from Example 14,Step A) was reacted with a preactivated DMA solution of 1.04 g (3.42mmol) of N-BOC-D-Tryptophan, 1.51 g of BOP, 0.46 g of HOBt, and 0.36 mLof NMM for 1.5 h. Washing and deprotection as per the general BOCprotocol above gave the title compound.

Step B: DβNal-(D-Trp)-Phe-(MBHA-resin)

The intermediate from step A was reacted with a preactivated DMAsolution of 1.08 g (3.42 mmol) of N-BOC-D-βnaphthylalanine, 1.51 g ofBOP, 0.46 g of HOBt, and 0.38 mL of NMM for 1.5 h. Washing anddeprotection as per the general BOC protocol above gave the titlecompound.

Step C: (inip)-DβNal-(D-Trp)-Phe-amide, TFA salt

The intermediate from step B was reacted with a preactivated DMAsolution of 0.78 g (3.42 mmol) of N-BOC-isonipecotic acid, 1.51 g ofBOP, 0.46 g of HOBt, and 0.38 mL of NMM for 2 h. Deblocking, washing,drying, and HF cleavage as per the general BOC protocol above gave 240mg of a powder. A 50 mg aliquot was purified by reverse phase HPLC(15-20 μ, 300 Å, Vydac C-18, 1×50 cm, gradient: 23-38% acetonitrile(0.1% TFA) in water (0.1% TFA) in 60 min at 9 mL/min, rt=30 min) to give23 mg of the title compound. MS (electrospray,M+H) 659.2.

EXAMPLE 16 ##STR65## (inip)-DβNal-DβNal-Phe, TFA salt

Step A: BOC-DβNal-Phe-(O-resin)

Commercially available N-BOC-Phe-(O-resin) (8.0 g, 0.55 mmol/g, 4.41mmol, 1.0 eq) was swelled in DCM, deblocked, washed, and coupled with2.78 g (8.82 mmol, 2.0 eq) of N-BOC-D-βnaphthylalanine using 7.8 g ofBOP, 1.2 G of HOBt, and 1.45 ml of NMM for 3 h, to giveN-BOC-DβNal-Phe-(O-resin) (ninhydrin negative) after washing.

Step B: BOC-DβNal-DβNal-Phe-(O-resin)

The above sample of BOC-DβNal-Phe-(O-resin) was deblocked, washed, andcoupled with 2.78 g (8.82 mmol, 2.0 eq) of BOC-D-β-naphthylalanine using7.8 g of BOP, 1.2 g of HOBt, and 1.45 ml of NMM for 3 h to giveBOC-DβNal-DβNal-Phe-(O-resin) (ninhydrin negative).

Step C: BOC-(inip)-DβNal-DβNal-Phe-(O-resin)

The above sample of BOC-DβNal-DβNal-Phe-(O-resin) was deblocked, washed,and coupled with 3.0 g (13.2 mmol, 3.0 eq) of N-BOC-isonipecotic acid(from Example 1, Method B, Step E) using 7.8 g of BOP, 1.2 g of HOBt,and 1.45 ml of NMM for 2 h to give BOC-(inip)-DβNal-DβNal-Phe-(O-resin)(ninhydrin negative), which was washed with methanol and dried in vacuo.Yield: 10.1 g

Step D: (inip)-DβNal-DβNal-Phe, TFA salt

A 3.0 g sample of the above BOC-(inip)-DβNal-DβNal-Phe-(O-resin) wasswelled in DCM, deblocked, washed with methanol, dried, and the productcleaved from the resin by the general HF procedure to give 790 mg of(inip)-DβNal-DβNal-Phe of approximately 90% purity. A 37 mg aliquot waspurified by HPLC (Vydac C-18, 27 to 42% acetonitrile in water over 60min, 0.1% TFA, 9 mL/min, 214 nm, rt=41-51 min) to give 4.3 mg of thetitle compound. MS (electrospray,M+H) 671.2.

EXAMPLE 17 ##STR66## (inip)-DβNal-DβNal-Phe methyl ester, TFA salt

To a solution of 50 mg of (inip)-DβNal-DβNal-Phe (from Example 16, StepD) in 20 ml of methanol was added 10 drops of 1 M HCl in diethyl ether.After stirring overnight, the reaction mixture was concentrated and theproduct purified by HPLC to give 22 mg of the title compound (VydacC-18, 1×50 cm, 9 mL/min, 27 to 42% acetonitrile in water over 60 min,0.1% TFA, 214 nm, rt=39-52 min). MS (electrospray,M+H) 685.4.

EXAMPLE 18 ##STR67## (inip)-DβNal-DβNal-(L-Phenylalanol), TFA salt

Step A: BOC-(inip)-DβNal-DβNal-(L-Phenylalanol)

To 50 ml of dry THF was added 1.5 g (ca. 0.8 mmol, 1.0 eq) ofBOC-(inip)-DβNal-DβNal- Phe-(O-resin) (from Example 16, Step C) and 4.0ml (80 mmol, 10 eq) of 2.0 M lithium borohydride solution in THF. Thereaction mixture was gently stirred for 1.5 h, 10 ml of HOAc addeddropwise, and stirring continued for another 0.5 h. The resin wasfiltered off, washed with methanol, and the combined filtrates partiallyevaporated. The product was partitioned between ethyl acetate and waterand the organic phase was washed with brine, dried, evaporated, and theproduct crystallized from ethyl acetate. IR (cm⁻¹): 3409, 3289, 3957,2930, 1695, 1635, 1536, 1164, 739, 699. MS (FAB, M+H) 757.4.

Step B: (inip)-DβNal-DβNal-(L-Phenylalanol), TFA salt

A solution of 50 mg of BOC-(inip)-DβNal-DβNal-L-Phenylalanol (step A) in2 ml of DCM was treated with 2 ml of TFA for 1 h, concentrated, and theproduct purified by HPLC (Vydac C-18, 1×50 cm, 31 to 45% acetonitrile inwater over 60 min, 0.1% TFA, 9 mL/min, 214 nm, rt=38-45 min). MS(electrospray,M+H) 657.4.

EXAMPLE 19 ##STR68## (inip)-DβNal-DβNal-(3(S)-3-benzyl-β-Ala) amide, TFAsalt

Step A: 3(S)-3-(t-Butoxycarbonylamino)-4-phenylpropionic acid(N-BOC-3(S)-3-benzyl-β-Ala)

This compound was most conveniently prepared by the method of Ondettiand Engel, J. Med. Chem. 18(7), (1975), 761-763. Using this procedure,2.0 g of commercially available N-BOC-L-Phenylalanine was converted to760 mg of the title compound in 36% overall yield. ¹ H NMR (300 MHz,CDCl₃) δ 11.3 (1H, s, exch), 7.2 (5H, m), 5.1 (1H, d), 2.85 (2H, m), 2.5(2H, m), 1.4 (9H, s). IR (cm⁻¹) 3316, 2977, 2930, 1709, 1662, 1496,1370, 1164, 1050, 1025, 746, 699. MS (FAB,M+H) 280.0.

Step B: BOC-(3(S)-3-benzyl-β-Ala)-(MBHA-resin)

A 2.54 g sample of MBHA-resin (0.64 mmol/g, 2.06 mmol) was swelled in1:1 DCM/DMA and coupled with 0.60 g (2.15 mmol, 1.1 eq) ofN-BOC-(3(S)-3-benzyl-β-Ala) (from Step A) using 1.44 g of BOP, 241 mg ofHOBt, and 196 μl of NMM for 72 h. the resin was washed and capped byacetylation with a mixture of acetic anhydride, TEA, and pyridine in DCMfor 15 min, giving the title compound after washing (ninhydrinnegative).

Step C: BOC-DβNal-(3(S)-3-benzyl-β-Ala)-(MBHA-resin)

The above sample of BOC-(3(S)-3-benzyl-β-Ala)-(MBHA-resin) wasdeblocked, washed, and coupled with N-BOC-D-β-napthylalanine using 2.85g of BOP, 290 mg of HOBt, and 708 μl of NMM for 2 h, according to thegeneral procedure, to give BOC-DβNal-(3(S)-3-benzyl-β-Ala)-(MBHA-resin)(ninhydrin negative).

Step D: BOC-DβNal-DβNal-(3(S)-3-benzyl-β-Ala)-MBHA-resin)

The above sample of BOC-DβNal-(3(S)-3-benzyl-β-Ala)-(MBHA-resin) wasdeblocked, washed, and coupled with N-BOC-D-β-naphthylalanine, using2.85 g of BOP, 290 mg of HOBt, and 708 μl of NMM for 2 h to give thetitle compound (ninhydrin negative).

Step E: BOC-(inip)-DβNal-DβNal-(3(S)-3-benzyl-β-Ala)-(MBHA-resin)

The product of Step D was deblocked, washed, and coupled withN-BOC-isonipecotic acid, using 2.85 g of BOP, 581 mg of HOBt, and 708 μlof NMM for 18 h to giveBOC-(inip)-DβNal-DβNal-(3(S)-3benzyl-β-Ala)-(MBHA-resin) (ninhydrinnegative).

Step F: (inip)-DβNal-DβNal-(3(S)-3benzyl-β-Ala)-amide, TFA salt

The above sample ofBOC-(inip)-DβNal-DβNal-(3(S)-3-benzyl-βAla)-(MBHA-resin) was deblocked,washed with methanol, and dried. The product was cleaved from the resinwith HF according to the general procedure to give 280 mg of a solid. A56 mg portion was purified by HPLC (Vydac C-18, 1×50 cm, 27 to 42%acetonitrile in water over 60 min, 9 mL/min, 0.1% TFA, rt=28-38 min) togive 23 mg of the title compound. MS(electrospray,M+H) 684.2.

EXAMPLE 20 ##STR69## (inip)-DβNal-DβNal-(N-2-phenylethyl-Gly) amide, TFAsalt

Step A: 2-Bromoacetyl-(MBHA-resin)

A 2.0 g sample of MBHA-resin (0.64 mmol/g, 1.28 mmol) was swelled in DCMand coupled with 0.36 g (2.56 mmol, 2.0 eq) of bromoacetic acid using3.84 ml (3.84 mmol, 3.0 eq) of 1M DIPC in DCM for 2 h. The resin waswashed with DCM (5×) to give 2-bromoacetyl-(MBHA-resin) (ninhydrinnegative).

Step B: BOC-DβNal-(N-2-phenylethyl-Gly)-(MBHA-resin)

The above sample of 2-Bromoacetyl-(MBHA-resin) was taken up in DCM and4.0 ml of phenethylamine added. After 5 h, the resin was washed(ninhydrin positive) and coupled with 806 mg (2.56 mmol, 2.0 eq) ofN-BOC-D-β-naphthylalanine using 1.70 g of BOP, 346 mg of HOBt, and 421μl of NMM for 18 h, to give the title compound (ninhydrin negative).

Step C: BOC-DβNal-DβNal-(N-2-phenylethyl-Gly)-(MBHA-resin)

The product of step B was deblocked, washed, and coupled to 806 mg (2.56mmol, 2.0 eq) of N-BOC-D-β-naphthylalanine using 1.70 g of BOP, 346 mgof HOBt, and 421 μl of NMM for 4 h to giveBOC-DβNal-DβNal-(N-2-phenylethyl-Gly)-(MBHA-resin) (ninhydrin negative).

Step D: BOC-(inip)-DβNal-DβNal-(N-2-phenylethyl-Gly)-(MBHA-resin)

The above sample of BOC-DβNal-DβNal-(N-2-phenylethyl-Gly)-(MBHA-resin)was deblocked, washed, and coupled to 586 mg (2.56 mmol, 2.0 eq) ofN-BOC-isonipecotic acid using 1.70 g of BOP, 346 mg of HOBt, and 421 μlof NMM for 4 h to giveBOC-(inip)-DβNal-DβNal-(N-2-phenylethyl-Gly)-(MBHA-resin) (ninhydrinnegative).

Step E: (inip)-DβNal-DβNal-(N-2-phenylethyl-Gly) amide, TFA salt

The resin from Step D was deblocked, washed with methanol, dried, andcleaved with HF according to the general procedure to give 802 mg of asolid. A 70 mg portion was purified by HPLC (Vydac C-18, 1×50 cm, 25 to40% acetonitrile in water over 60 min, 0.1% TFA, 9 mL/min, rt=33-50 min)to give 42 mg of the title compound. MS (electrospray,M+H) 684.2.

EXAMPLE 21 ##STR70## (inip)-DβNal-DβNal-(3-I-Tyr), TFA salt Step A:[3-I-Tyr(3-BrBzl)]-(O-resin)

N-BOC-(O-3-Bromobenzyl)-3-Iodo-L-Tyrosine [Peninsula Labs,BOC-(3-I-Tyr(3-BrBzl)] was coupled to Hydroxymethyl-resin (Bachem, 1%DVB, 100-200 mesh, 1.0 mmol/g) with DIPC (3 eq) and DMAP (0.25 eq) inDMA for 3 h. The resin was washed, deblocked, and washed again,according to the general procedure to give the title compound (ninhydrinpositive).

Step B: DβNal-[3-BrBzl)]-(O-resin)

BOC-DβNal (SyntheTech, 3 eq) was activated with HTU (RichelieuBiotechnologies, 4 eq) and DIPEA in DMA and coupled to the resin for 1 h(ninhydrin negative). The resin was washed, deblocked, and washed again,to give the title compound (ninhydrin positive).

Step C: DβNal-DβNal-[3-BrBzl)]-(O-resin)

BOC-DβNal (3 eq) was activated with HBTU (4 eq) and DIPEA in DMA andcoupled to the resin for 1 h (ninhydrin negative). The resin was washed,deblocked, and washed again, to give the title compound (ninhydrinpositive).

Step D: (inip)-DβNal-DβNal-[3-I-Tyr(3-BrBzl)]-(O-resin)

N-BOC-isonipecotic acid (3 eq) was activated with HBTU (4 eq) and DUPEAin DMA and coupled to the above resin for 1 h (ninhydrin negative). Theresin was washed, deblocked, washed with DCM, MeOH, and dried in vacuoto give the title compound (ninhydrin positive).

Step E: (inip)-DβNal-DβNal-(3-I-Tyr), TFA salt

The intermediate from step D was cleaved with anhydrous HF according tothe general procedure to give 350 mg of a solid, which was purified byreverse phase HPLC (15-20μ, 300 Å, Vydac C-18, 2.5×27 cm, gradient:32-46% acetonitrile (0.1% TFA) in water (0.1% TFA) in 80 min at 18mL/min, rt=20 min) to give 101 mg of (inip)-DβNal-DβNal-(3-I-Tyr), TFAsalt as a colorless powder after lyophilization. MS (electrospray, M+H)813.0.

EXAMPLE 22 ##STR71## (inip)-DβNal-DβNal-[N-(2-phenylethyl),N-(4-aminobutyl)]amide, TFA salt

Step A: DβNal-[4-N-(2-phenylethyl)]-(BDA-COO-resin)

A 1.0 g sample of the BDA-COO-resin from (Example 13, Step A) wasswelled with DCM, and a solution of 1% HOAc in DMF added, followed by 69mg (1.2 eq) of phenylacetaldehyde and 60 mg of sodium cyanoborohydride.After 14 h, the resin was washed repeatedly with DMA and DCM (ninhydrintest showed a red color replacing the deep blue of the starting resin),and the resin neutralized. To a DCM slurry of the resin was added 0.45 g(1.44 mmol) of BOC-D-β-naphthylalanine, 0.43 g of BOP-Cl, and 0.30 mL ofDIPEA. After 14 h, the resin was washed, deblocked, and washed again togive the title compound (ninhydrin positive).

Step B: DβNal-DβNal-[4-N-(2-phenylethyl)]-(BDA-COO-resin)

The intermediate from step A was reacted with a preactivated DMAsolution of 0.45 g (1.44 mmol) of BOC-D-β-naphthylalanine, 0.64 g ofBOP, 0.19 g of HOBt, and 0.21 mL of NMM for 1.5 h. The resin was washed,deblocked, and washed again, giving the title compound (ninhydrinnegative).

Step C: (inip)-DβNal-DβNal-[N-(2-phenylethyl), N-(4-aminobutyl)]amide,TFA salts

The intermediate from step B was reacted with a preactivated DMAsolution of 0.33 g (1.44 mmol) of N-BOC-isonipecotic acid, 0.64 g ofBOP, 0.19 g of HOBt, and 0.21 mL of NMM for 2 h. The resin was washed,deblocked, washed with methanol, and dryed in vacuo. HF cleaveage as perthe general BOC protocol above gave 100 mg of a powder, that waspurified by reverse phase HPLC (15-20μ, 300 Å, Vydac C-18, 1×50 cm,gradient: 23-38% acetronitrile (0.1% TFA) in water (0.1% TFA) in 60 minat 9 mL/min. rt=35 min) to give 10 mg of the title compound. MS(electrospray, M+H) 698.4.

EXAMPLE 23 ##STR72## (inip)-DβNal-DβNal-(N-2-phenylethyl)amide, TFA salt

Step A: FMOC-DβNal-(Wang resin)

Wang resin (6.0 g, 0.63 mmol/g, 3.78 mmol) was swelled in DCM andcoupled with 2.48 g (5.67 mmol) of N-FMOC-D-β-naphthylalanine using DIPC(11.3 mL of a 1 M solution in DCM, 11.3 mmol) and 100 mg of DMAP in DCMfor 6 h, to give the title compound, after washing with methanol anddrying in vacuo (yield 8.14 g).

Step B: Dβ-Nal-DβNal-(Wang resin)

Employing the standard FMOC chemistry cycle above, a 4.0 g sample ofFMOC-DβNal-(Wang resin) (0.63 mmol/g,2.52 mmol) was swelled in DCM,deblocked, washed, and coupled with 2.20 g (5.04 mmol) of N-FMOC-DβNalusing 2.23 g of BOP, and 0.83 ml of NMM for 1.5 h. The sample was thendeblocked and washed to give DβNal-DβNal-(Wang resin) (ninhydrinpositive).

Step C: BOC-(inip)-Dβ-Nal-DβNal-(Wang resin)

The above sample of Dβ-Nal-DβNal-(Wang resin) was coupled with 2.31 g(10.1 mmol) of N-BOC isonipecotic acid (from Example 1, Method B, StepE) using 4.45 g of BOP and 1.38 ml of NMM for 2 h to giveBOC-(inip)-Dβ-Nal-(Wang-resin) (ninhydrin negative), which was washedwith methanol and dried in vacuo.

Step D: (inip) NβNal-DβNal-(N-2-phenylethyl) amide, TFA salt

A 0.30 g sample of the above BOC-(inip)-DβNal-(Wang resin) was suspendedin 5 mL of phenethylamine (Aldrich) and 5 mL of DMA. The stirred mixturewas placed in a 50° C. oil bath under nitrogen for 18 h, filtered, andthe resin washed with DCM. The filtrate was concentrated and partitionedbetween 1 N sodium hydrogen sulfate and ethyl acetate. The organic phasewas washed successively with 1 N sodium bicarbonate, water, brine, anddried over magnesium sulfate. Concentration gave an oil that was treatedwith 6 mL of DCM/TFA (1:1) for 1 h at ambient temperature andconcentrated to give 30 mg of a gum. Purification by reverse phase HPLC(15-20μ, 300 Å, Vydac C-18, 1×50 cm, gradient: 23-38% acetonitrile (0.1%TFA) in water (0.1% TFA) in 60 min at 9 mL/min, rt=33 min) gave 7.6 mgof the title compound. MS (electrospray, M+H) 627.6.

EXAMPLE 24 ##STR73## (inip)-DβNal-DβNal-[N-(4-aminobutyl)]amide, TFAsalt

Step A: BOC-DβNal-(BDA-COO-resin)

A 5.0 g (0.24 mmol/g, 1.2 mmol) sample of the BDA-COO-resin from Example13, Step A was neutralized, washed with DCM, and coupled with 0.76 g(2.4 mmol) of BOC-Dβ-Nal, 1.06 g of BOP, 0.32 g of HOBt, and 0.40 mL ofNMM for 2 h according to the general BOC chemistry protocols givenabove. The resin was washed, giving BOC-Dβ-Nal-(BDA-COO-resin)(ninhydrin negative).

Step B: BOC-DβNal-DβNal-(BDA-COO-resin)

The above sample was deblocked, washed, neutralized, washed, and coupledwith 0.76 g (2.4 mmol) of BOC-Dβ-Nal, 1.06 g of BOP, 0.32 g of HOBt, and0.40 mL of NMM for 2 h, giving BOC-DβNal-DβNal-(BDA-COO-resin)(ninhydrin negative).

Step C: (inip)-DβNal-DβNal-(BDA-COO-resin)

The resin from Step B was washed, deblocked, washed, neutralized,washed, and coupled with 0.55 g (2.4 mmol) of N-BOC-isonipecotic acid(Example 1, Method B, step E), 1.06 g of BOP, 0.32 g of HOBt, and 0.40mL of NMM for 2h, giving BOC-(inip)-DβNal-DβNal-(BDA-COO-resin)(ninhydrin negative). The resin was washed with DCM, deblocked, washedwith DCM, methanol, and dried in vacuo.

Step D: (inip)-DβNal-DβNal-[N-(4-aminobutyl)]amide, TFA salt

The above resin (6 g) was cleaved with HF according to the generalprocedure and afforded 320 mg of a solid after lyophilization. Thesample was purified by reverse phase HPLC (15-20μ, 300 Å, Vydac C-18,1×50 cm, gradient: 12%-26% acetronitrile (0.1% TFA) in water (0.1% TFA)in 80 min at 9 mL/min, rt=18 min) to give 138 mg of the title compound.MS (electrospray, M+H) 594.2.

EXAMPLE 25 ##STR74## (inip)-DβNal-(N-Me-DβNal)-[N-4-aminobutyl)]amide,TFA salt

Step A: BOC-(N-Me-DβNal)-(BDA-COO-resin)

A 1.0 g sample of BDA-COO-resin from Example 13, Step A was washed,neutralized with 5% DIPEA/DCM, washed, and coupled with 981 mg (3 mmol)of BOC-(N-Me-DβNal), 1.32 g of BOP, and 0.45 mL of NMM for 1 h givingBOC-(N-Me-DβNal)-BDA-COO-resin) (ninhydrin negative).

Step B: BOC-DβNal-(N-Me-DβNal)-(BDA-COO-resin)

The above sample was washed, deblocked, washed, neutralized with 5%DIPEA/DCM, and washed. BOC-DβNal (3.29 g, 10 mmol) was activated withDIPC (5.0 mL, 1.0 M in DCM) in 15 mL DCM for 4 min, then added to theresin. After 6 h, the resin was washed, givingBOC-DβNal-(N-Me-DβNal)-(BDA-COO-resin) (ninhydrin negative).

Step C: (inip)-DβNal-(N-Me-DβNal)-(BDA-COO-resin)

The above sample was deblocked, washed, neutralized with 5% DIPEA/DCM,washed, and coupled with 648 mg (3 mmol) of N-BOC-isonipecotic acid,1.32 g of BOP, and 0.45 mL of NMM for 1 h, givingBOC-(inip)-DβNal-(N-Me-DβNal)-(BDA-COO-resin) (ninhydrin negative). Theresin was washed with DCM, deblocked, washed with DCM, methanol, anddried in vacuo to give 1.2 g of the title compound.

Step D: (inip)-DβNal-(N-Me-DβNal)-[N-(4-aminobutyl)]amide, TFA salt

The above resin (1.2 g was cleaved with HF according to the generalprocedure to afford 100 mg of a solid after lyophilization. A 57 mgsample was purified by reverse phase HPLC (15-20μ, 300 Å, Vydac C-18,1×50 cm, gradient: 20-35% acetonitrile (0.1% TFA) in water (0.1% TFA) in60 min at 9 mL/min, rt=28 min) to give 27 mg of the pure title compound.MS electrospray, M+H) 607.7.

EXAMPLE 26 ##STR75## (inip)-DβNal-(N-Me-DβNal)-N-(4-piperidinyl) amide,TFA salt

Step A: N-CBZ-isonipecotic acid

Benzyl chloroformate (16.4 mL, 115 mmol) in toluene (50 mL) was addeddropwise to a stirred solution of 12.9 g (100 mmol) of isonipecotic acid(Aldrich) and 21.0 g (250 mmol) of sodium bicarbonate in 200 mL ofwater. After 14 h, the mixture was extracted with ether (3×50 ml) andthe ether layers were discarded. The aqueous layer was acidified withconc. HCl to pH 2, causing the product to precipitate. The product waspartitioned into ethyl acetate (3×50 mL) and the combined organic layerswere washed with brine, dried over magnesium sulfate, and concentratedin vacuo to yield 22.6 g (86%) of N-CBZ-isonipecotic acid as a viscousoil.

Step B: N-CBZ-4-(BOC-amino)-piperidine

A solution of N-CBZ-isonipecotic acid (10.3 g, 38.9 mmol) in tert-butylalcohol (100 mL) and DCM (100 mL) was treated with diphenylphosphorylazide (11.8 g, 42.8 mmol), TEA (5.97 mL, 42.8 mmol), and the resultingmixture was heated at reflux for 3 days. The solution was concentratedin vacuo and the residue was partitioned between ether and water. Theorganic layer was washed successively with 10% aq citric acid, sat.sodium bicarbonate, brine, dried over magnesium sulfate, andconcentrated to an oil. This residue was purified by silica gel flashchromatography (gradient elution, 7:3 to 1:1 hexane-ether) to afford 3.2g (25%) of the title compound as a colorless crystalline solid: TLCR_(f) 0.21 (1:1 hexane/ethyl ether).

Step C: 4-(BOC-amino)-piperidine

N-CBZ-4-(BOC-amino)-piperidine (3.0 g, 9.0 mmol) was dissolved inethanol (100 mL) and transferred into a Parr shaker bottle. After adding10% palladium on carbon (0.5 g), the mixture was shaken under anatmosphere of hydrogen at 50 psi for 0.75 h on a Parr apparatus. Thecatalyst was removed by filtration through a pad of Celite. The filtercake was washed with ethanol and the combined filtrate and washings wereconcentrated in vacuo to yield 1.8 g (100%) of crude4-(BOC-amino)-piperidine as a pale yellow oil. This product was usedimmediately in the next step without further purification.

Step D: [4-(BOC-amino)-piperidine]-(COO-resin)

Hydroxymethyl resin (4.0 g of 0.45 mmol/g, 1.8 mmol) was rinsed severaltimes with tolune. A solution of 20% phosgene in toluene (50 mL) wasadded to the hydroxymethyl resin (2×30 min) to generate thechloroformate intermediate. After rinsing the resin several times withtoluene and dioxane, a solution of 4-(BOC-amino)-piperidine (Step C, 1.8g, 9.0 mmol) in dioxane was added, and the resulting mixture wasagitated for 3 h. The resin was rinsed with dioxane, DCM, and dried invacuo, to provide 4.4 g of the title compound.

Step E: BOC-(N-Me-DβNal)-[4-(4-amino-piperidine)]-(COO-resin)

An aliquot (0.82 g, ˜0.33 mmol) of the resin from Step D was treatedwith TFA deblock, neutralized, washed, and coupled with 3 eq ofBOC-N-Me-DβNal) (from Example 4, Step B), 3 eq of BOP, 3 eq of HOBt and4.5 eq of NMM in DMA/DCM for 1 h, after which a negative ninhydrin testwas observed.

Step F: FMOC-DβNal-(N-Me-DβNal)-[4-(4-amino-piperidine)]-(COO-resin)

The above sample of BOC-(N-Me-DβNal)-[4-(4-amino-piperidine)]-(COO-resin) was deblocked with TFA, neutralized,washed, and coupled with 4 eq of FMOC-D-β-naphthylalanine, 4 eq ofBOP-Cl, and 6 eq of DIPEA in DCM overnight, after which a negativeninhydrin test was observed.

Step G: (inip)-DβNal-(N-Me-DβNal)-N-(4-piperidinyl) amide, TFA salt

The above sample ofFMOC-DβNal-(N-Me-DβNal)-[4-(4-amino-piperidine)]-(COO-resin) wasdeblocked with 20% piperidine/DMA, washed, and coupled with 3 eq ofN-BOC-isonipecotic acid (from Example 1, Method B, Step E), 3 eq of BOP,3 eq of HOBt, and 4.5 eq of NMM in DMA/DCM for 1 h, after which anegative ninhydrin test was observed. The peptide was deblocked withTFA, washed, and dried in vacuo to give(inip)-DβNal-(N-Me-DβNal)-[4-(4-amino-piperidine)]-(COO-resin). Thispeptide was cleaved from the resin with HF and lyophilized as per thegeneral procedure to provide 88 mg of a crude solid. This solid waspurified by reverse phase HPLC (15-20μ, 300 Å, Vydac C-18, 1×50 cm,gradient: 23-38% acetonitrile (0.1% TFA) in water (0.1% TFA) in 60 minat 9 mL/min, rt=43 min) to give 39 mg of the title compound as acolorless powder after lyophilization. MS (electrospray, M+H) 619.4.

EXAMPLE 27 ##STR76## (inip)-DβNal-(D-β-napthylalanol), TFA salt

A 0.30 g sample of BOC-(inip)-DβNal-DβNal-(Wang resin) (from Example 23,Step C) was suspended in 5 mL of THF under nitrogen and 0.90 mL of a 2.0M solution of lithium borohydride in THF (Aldrich) added. After 1.5 h, 2mL of HOAc was added carefully, the suspension filtered, and the resinwashed with MeOH. The combined filtrates were concentrated three timesfrom MeOH to give a solid, which was treated with 9 mL of TFA/DCM (3:1)containing a few drops of triethylsilane for 1 h. The solution wasconcentrated to give 270 mg of a solid, which was purified by reversephase HPLC (15-20μ, 300 Å, Vydec C-18, 2.5×27 cm, gradient: 25-39%acetonitrile (0.1% TFA) in water (0.1% TFA) in 80 min at 18 mL/min,rt=50 min) to give 39 mg of the title compound. MS (electrospray, M+H)510.0.

EXAMPLE 28 ##STR77## (inip)-DβNal-(N-Methyl-D-β-naphthylalanol), TFAsalt

Step A: (N-Me-DβNal)-(O-resin)

A 1.48 g (4.50 mmol) sample of BOC-N-Methyl-D-β-naphthylalanine (fromExample 4, Step B) was coupled to 1.5 g (1.0 mmol/g, 1.5 mmol) ofhydroxymethyl-resin with DIPC (4.50 mL of a 1.0 M solution in DCM, 4.50mmol) and 55 mg (0.45 mmol) of DMAP in DMA/DCM (1:1) for 2 h. The resinwas washed, deblocked, and washed according to the general BOCprocedure, to give the title compound (beads give an orange ninhydrintest).

Step B: DβNal-(N-Me-DβNal)-(O-resin)

The above (D-Me-DβNal)-(O-resin) was coupled with 1.42 g (4.50 mmol) ofBOC-D-β-naphthylalanine, 1.01 g of BOP-Cl, and 1.46 mL of DIPEA in DCMfor 12 h. The resin was washed and deblocked (ninhydrin positive) togive the title compound.

Step C: BOC-(inip)-DβNal-(N-Me-DβNal)-(O-resin)

To a slurry of the above DβNal-(N-Me-DβNal)-(O-resin) in DCM/DMA (1:1)was added 1.03 g (4.50 mmol) of N-BOC-isonipecotic acid, 1.99 g of BOP,and 0.61 g of HOBt, followed by 1.0 mL of NMM. After 1 h, the resin waswashed, washed again with methanol, and dried in vacuo to give 2.33 g ofthe title compound.

Step D: (inip)-DβNal-(N-Methyl-D-β-naphthylalanol), TFA salt

A 1.0 g (0.64 mmol) sample of the aboveBOC-(inip)-DβNal-(n-Me-DβNal)-(O-resin) was suspended in 10 mL of THFunder nitrogen and 3.2 mL of a 2.0 M solution of lithium borohydride inTHF (Aldrich) added. After 1.5 h, 2 mL of HOAc was added carefully, thesuspension filtered, and the resin washed with MeOH. The combinedfiltrates were concentrated 3× from MeOH to give a solid, which wastreated with 6 mL of TFA/DCM (1:1), containing a few drops oftriethylsilane, for 1 h. The solution was concentrated to give 700 mg ofa solid containing salts. A 386 mg aliquot was purified by reverse phaseHPLC (15∝20μ, 300 Å, Vydac C-18, 1×50 cm, gradient: 23-38% acetonitrile(0.1% TFA) in water (0.1% TFA) in 60 min at 9 mL/min, rt=40 min) to give21 mg of the title compound. MS (electrospray, M+H) 525.0.

EXAMPLE 29 ##STR78## BOC-(inip)-DβNal-{N-methyl,N-[(2R)-2-(1-amino-3-(2-naphthyl)propyl]}amide, TFA salt

Step A: BOC-(inip)-DβNal

To a stirred solution of 10.0 g (44.0 mmol, 1.0 eq) ofN-BOC-isonipecotic acid (from Example 1, Method B, Step E) in 200 ml ofDCM was added 100 mg of DMAP and 4.53 g (22 mmol, 0.5 eq) of DCC. After1 h, the dicyclohexylurea was filtered off and the filtrate added to asolution of 4.7 (22.0 mmol, 0.5 eq) of D-β-naphthyalanine and 35 ml ofNMM in 200 ml of DCM. The reaction was stirred overnight, concentrated,and the residue partitioned between ethyl acetate and 0.5 N citric acid.The organic phase was washed with water, brine, evaporated, and theproduct recrystallized from ethyl acetate, to give 3.95 g ofBOC-(inip)-DβNal. Another 2.14 g was obtained by chromatography of themother liquors on silica (ethyl acetate/HOAc, 98:2), combined yield:65%. ¹ H NMR (300 MHz, d₆ -acetone) δ7.8 (3H, m), 7.65 (1H, s), 7.4 (3H,m), 7.2 (1H, d), 4.8 (1H, m), 3.88 (2H, m), 3.35 (1H, m), 3.15 (1H, m),2.62 (2H, m), 2.35 (1H, m), 1.6-1.4 (4H, m), 1.35 (9H, s). MS (FAB, M+H)427.2.

Step B: BOC-(N-Me-D-β-naphthylalanol)

To a cold solution of 3.25 g (9.9 mmol, 1.0 eq) of BOC-(N-Me-DβNal)(from Example 4, Step B) and 1.10 g (10.9 mmol, 1.1 eq) of TEA in 50 mlof dry THF, was added dropwise over 30 min, a solution of 1.19 g (10.9mmol, 1.1 eq) of ethyl chloroformate in 10 ml of dry THF. The reactionturned bright red and TEA hydrochloride precipitated and was filteredoff. The red filtrate was added dropwise to a cold, stirred solution of1.50 g (39.6 mmol, 4.0 eq) of sodium borohydride in 50 ml ofmethanol/water (1:1). After stirring overnight, the reaction mixture wasconcentrated to 1/2 the initial volume and partitioned between ethylacetate and 0.5 N citric acid. The organic phase was washed successivelywith water, 10% potassium carbonate, saturated sodium bicarbonate,brine, dried over sodium sulfate, filtered, and concentrated to yield2.91 g of an oil (94%). ¹ H NMR (300 MHz, CDCl₃) δ 7.75 (3H, m), 7.6(1H, s), 7.4 (3H, m), 4.3 (1H), 3.7 (2H, m), 3.0-2.8 (6H, m), 1.3 (9H,2s). IR (cm⁻¹) 3428, 3050, 2977, 1689, 1669, 1363, 1144. MS (EI, e/m)427.2.

Step C: (2R)-1-Azido-2-BOC-methylamino)-3-(2-naphthyl) propane

A cold solution of hydrazoic acid (4.14 mmol, 1.5 eq) in benezene wasprepared by acidification of a stirred mixture of 269 mg (4.14 mmol) ofsodium azide, 5 ml of water and 5 ml of benzene with 5 ml of 3.6 Nsulfuric acid at 10° C. The benzene phase was separated, dried withsodium sulfate, and filtered. This solution was added to a stirredmixture of 867 mg (3.31 mmol, 1.2 eq) of triphenylphosphine and 669 mgof diisopropylazodicarboxylate (3.31 mmol, 1.2 eq) in dry THF at -78° C.A solution of 870 mg (2.76 mmol, 1.0 eq) of BOC-(N-Me-D-β-naphthylalanol(step B) in 10 ml of dry THF was added and the reaction mixture allowedto come to room temperature over 2 h. Saturated sodium bicarbonate (20mL) was added and the reaction mixture partially concentrated to removethe THF. The mixture was partitioned between ethyl acetate and saturatedsodium bicarbonate and the organic phase was washed with brine, driedover sodium sulfate, filtered, and evaporated. Flash chromatography onsilica (hexane/ethyl acetate, 80:20) gave 600 mg (64%) of the titlecompound. ¹ H NMR (300 MHz, CDCl₃) δ 7.75 (3H, m), 7.6 (1H, d), 7.42(2H, m), 7.25 (1H, m), 4.4 (1H, m), 3.6-2.8 (4H, m), 2.7 (3H, 2s), 2.3(9H, 2s). IR (cm⁻¹) 3057, 2977, 2094, 1689.

Step D: BOC-(inip)-DβNal-{N-methyl,N-[(2R)-2-(1-azido-3-(2-naphthyl)propyl]}amide

A solution of 300 mg of (2R)-1-azido-2-(BOC-methylamino)-3-(2-naphthyl)propane (0.88 mmol. 1.0 eq) (step C) in 2.0 ml of DCM was treated with2.0 ml of TFA for 1 h then evaporated several times from DCM. Theresidue was dissolved in a solution of 5 ml of DCM and 250 μl of NMMthen added to a previously prepared mixture of 751 mg (1.76 mmol, 2.0eq) of N-BOC-(inip)-DβNal (step A), 363 mg (1.76 mmol, 2.0 eq) of DCC,238 mg (1.76 mmol, 2.0 eq) of HOBt, and 250 μl of NMM in 15 ml ofDCM/DMF (2:1). The reaction was stirred overnight, triturated with 1 mLof water and the precipitated dicyclohexylurea was filtered off. Thereaction mixture was taken up in ethyl acetate and washed successivelywith 0.5 N citric acid, water, 10% potassium carbonate, water, saturatedsodium bicarbonate, brine, dried over sodium sulfate, filtered, andevaporated. Flash chromatography on silica (ethyl acetate/hexane (1:1),Rf=0.4) gave 380 mg (67%) of the title compound. IR (cm⁻¹) 3309, 3057,2977, 2930, 2100, 1689, 1635, 1171. MS (FAB, M+H) 649.4.

Step E: BOC-(inip)-DβNal-{N-methyl,N-[(2R)-2-(1-amido-3-(2-naphthyl)propyl]}amide, TFA salt

A solution of 280 mg (0.43 mmol) of the azine from step D in 15 ml ofmethanol and 1 ml HOAc was hydrogenated over 100 mg of 10% palladium oncarbon at 30 psi for 8 hrs. The catalyst was filtered off and thefiltrate evaporated to give 250 mg of crude product. A 20 mg aliquot waspurified by reverse phase HPLC (15-20μ, 300 Å, Vydac C-18, 1×50 cm,gradient: 30-45% acetonitrile (0.1% TFA) in water (0.1% TFA) in 60 minat 9 mL/min, rt=39-48 min) to give 8 mg of the title compound. IR (cm⁻¹)3409, 3296, 3057, 2977, 1675, 1430, 1204, 1171, 1131. MS (electrospray,M+H) 624.5.

EXAMPLE 30 ##STR79## (inip)-DβNal-{N-methyl,N-[(2R)-2-(1-amino-3-(2-naphthyl)propyl]}amide, TFA salt

A solution of 200 mg of BOC-(inip)-DβNal-{N-methyl,N-[(2R)-2-(1-amino-3-(2-naphthyl)propyl]} amide, TFA salt (from Example29, step E) in 2 ml of DCM was treated with 2 ml of TFA, stirred for 1h, and concentrated to give 490 mg of crude product. A 100 mg aliquotwas purified by HPLC (Vydac C-18, 1×50 cm, 20 to 35% acetonitrile inwater, 60 min, 0.1% TFA, 9 mL/min, rt=21-31 min) giving 17 mg of thetitle compound. MS (electrospray, M+H) 523.2.

EXAMPLE 31 ##STR80## (inip)-DβNal-{N-methyl,N-[(2R)-2-(1-acetamido-3-(2-naphthyl)propyl]}amide, TFA salt

To a solution of 33 mg (0.05 mmol) of BOC-(inip)-DβNal-{N-methyl,N-[(2R)-2-(1-amino-3-(2-naphthyl)propyl]} amide, TFA salt (from Example29, step E) in 0.5 ml of DCM was added 0.5 ml of TEA and 0.5 ml ofacetic anhydride. The reaction mixture was stirred for 1 h thenevaporated and partitioned between ethyl acetate and water. The organicphase was dried over sodium sulfate and evaporated. The residue wastaken up in 2 ml of DCM and 2 ml of TFA added. The mixture was stirredfor 1 h, evaporated and the crude product (48 mg) was purified by HPLC(Vydac C-18, 1×50 cm, 25 to 50% acetonitrile in water over 60 min, 9mL/min, 0.1% TFA, rt=29 min) giving 15 mg of the title compound. MS(electrospray, M+H) 565.4.

EXAMPLE 32 ##STR81## (inip)-DβNal-[N-1-{2-(2-naphthyl)ethyl}]amide, TFAsalt

Step A: 2-(2-Naphthyl)ethylamine hydrochloride

A solution of lithium aluminum hydride (LAH, 100 mmol, 3.0 eq) in 300 mlof dry ether at 0° C. was stirred and a solution of2-naphthylacetonitrile (5.0 g, 30 mmol, 1.0 eq) in 200 ml of dry etheradded over 2 h. To the resultant bright orange slurry, was addeddropwise, 25 ml of cold (0° C.) 12 N sulfuric acid, and the mixturestirred until colorless. The reaction mixture was partitioned betweenether and water and the ether phase, containing mostly unreacted2-naphthylacetonitrile, discarded. The aqueous phase was basified withsodium hydroxide and the separated free amine extracted into ether,dried over sodium sulfate, filtered, and acidified with anhydrous HCl indioxane. The precipitated HCl salt was collected by filtration to give890 mg of the title compound. ¹ H NMR (300 MHz, D₂ O) δ 7.82 (3H, t),7.7 (1H, s), 7.45 (2H, m), 7.38 (1H, d) 3.25 (2H, t), 3.05 (2H, t). MS(FAB), M+H) 172.1.

Step B: BOC-(inip)-DβNal-[N-1-{2-(2-naphthyl)ethyl}]amide

A mixture of 100 mg (0.23 mmol, 1.0 eq) of N-BOC-(inip)-DβNal (fromExample 29, step A), 42 mg (0.23 mmol, 1.0 eq) of2-(2-naphthyl)ethylamine hydrochloride (step A), 132 mg (0.69 mmol, 3.0eq) of EDC, 31 mg (0.23 mmol, 1.0 eq) of HOBt, 87 μl (0.69 mmol, 3.0 eq)of NMM, and 5.0 ml of DMF was stirred overnight at ambient temperature.The reaction mixture was partitioned between ethyl acetate and dilutehydrochloric acid and the separated organic phase washed successivelywith water, saturated sodium bicarbonate, brine, dried over sodiumsulfate, filtered, and evaporated. The crude product was chromatographedon silica (ethyl acetate/hexane (70:30), Rf=0.5) to give 110 mg of thetitle compound. IR (cm⁻¹) 3289, 3057, 2977, 2930, 2857, 1695, 1642,1423, 1171, 819, 739. MS (FAB, M+H) 580.3.

Step C: (inip)-DβNal-[N-1-{2-(2-naphthyl)ethyl}]amide, TFA salt

A solution of 110 mg ofN-BOC-(inip)-DβNal-[N-1-{2-(2-naphthyl)ethyl}]amide (step B) in 4 ml ofDCM was treated with 2 ml of TFA and stirred for 2 h. The concentratedcrude product (121 mg) was purified by HPLC (1×50 cm, Vydac C-18, 25 to40% acetonitrile in water over 60 min, 9 mL/min, 0.1% TFA, rt=25-30 min)to give 29 mg of the title compound. MS (electrospray, M+H) 479.8.

EXAMPLE 33 ##STR82## (inip)-DβNal-[N-methyl,N-1-{2-(2-naphthyl)ethyl}]amide, TFA salt

Step A: N-Methyl-N-2-(2-naphthyl)ethylamine, TFA salt

A solution of 2-(2-naphthyl)ethylamine (1.4 mmol, 1.0 eq) in DCM wasprepared by partitioning 250 mg of 2-(2-naphthyl)ethylaminehydrochloride (from Example 32, step A) between 5 ml of DCM and 5 ml of10% aq sodium hydroxide. The organic phase was dried over sodiumsulfate, filtered, and 363 mg (1.67 mmol, 1.2 eq) ofdi-t-butyldicarbonate added, followed by 1.0 ml of TEA. After stirringfor 30 min, the reaction mixture was concentrated and the productcrystallized from hexane at -78° C.

Yield: 250 mg.

This product was dissolved in 10 ml of dry THF, 262 μl (4.2 mmol, 3.0eq) of methyl iodide added, and the solution cooled to 0° C. Sodiumhydride (47 mg, 60% dispersion in mineral oil, 1.96 mmol) was added inportions with stirring over 10 min, and the reaction allowed to warm toambient temperature overnight. The solvents were evaporated and thecrude product taken up in ethyl acetate/hexane and washed successivelywith saturated sodium bicarbonate, 1 N sodium bisulfite, water, brine,dried over sodium sulfate, filtered, and evaporated. Treatment withTFA/DCM (1:1) for 1 h and concentration gave 190 mg of the titlecompound as an oil. ¹ H NMR (300 MHz, CDCl₃) δ 7.78 (3H, t), 7.62 (1H,s), 7.4 (2H, m), 7.32 (1H, d), 2.9 (4H, m), 2.4 (3H, s), 1.55 (1H, s).MS (FAB, M+H) 186.0.

Step B: BOC-(inip)-DβNal-[N-methyl, M-1-{2-(2-naphthyl)ethyl}]amide

A mixture of 525 mg (1.23 mmol, 1.2 eq) of BOC-(inip)-DβNal (fromExample 29, step A), 190 mg (1.02 mmol, 1.0 eq) ofN-methyl-N-2-(2-naphthyl)ethylamine (step A), 584 mg (3.06 mmol, 3.0 eq)of EDC, 138 mg (1.02 mmol, 1.0 eq) of HOBt, 412 mg of (4.08 mmol, 4.0eq) of NMM, and 15 ml of DMF was stirred overnight at ambienttemperature. The reaction mixture was partitioned between ethyl acetateand dilute citric acid and the separated organic phase washedsuccessively with water, saturated sodium bicarbonate, brine, dried oversodium sulfate, filtered, and evaporated. The crude product waschromatographed on silica (80:20, ethyl acetate/hexane) to give 550 mgof the title product. IR (cm⁻¹) 3302, 3050, 2977, 2930, 1689, 1629,1423, 1164, 819, 732.

Step C: (inip)-DβNal-[N-methyl, M-1-{2-(2-naphthyl)ethyl}]amide, TFAsalt

A solution of 550 mg of BOC-(inip)-(inip)-DβNal-[N-methyl,M-1-{2-(2-naphthyl)ethyl}]amide (step B) in 4 ml of DCM was treated with3 ml of TFA, stirred for 2 h, and concentrated to give 569 mg of crudeproduct. A 90 mg aliquot was purified by HPLC (1×50 cm, Vydac C-18, 27to 42% acetonitrile in water over 60 min, 0.1% TFA, 9 mL/min, 214 nm,rt=30-45 min) to give 29 mg of the title compound. MS (electrospray,M+H) 493.8.

EXAMPLE 34 ##STR83## (inip)-DβNal-(N-(2-naphthyl)methyl) amide, TFA salt

Step A: 2-Aminomethylnaphthylene hydrochloride

A stirred 0° C. solution of 20.0 g of 2-naphthaldehyde (128 mmol) and98.7 g (1.28 mol) of ammonium acetate in 200 mL of MeOH/HOAc (99:1) wastreated with 5.62 g (90.0 mmol) of sodium cyanoborohydride, portionwise.The solution was stirred at ambient temperature for 24 h, concentratedin vacuo, resuspended in water, and basified with sodium hydroxide. Theproduct was extracted into ether, washed with water, brine, dried overmagnesium sulfate, and filtered. The filtrate was treated with a dryethereal solution of HCl, and the precipitated product washed with etherand dried to give 16.5 g (65% of 2-aminomethylnaphthylene hydrochloride.

Step B: (inip)-DβNal-(2-aminomethylnaphthyl) amide, TFA salt

A mixture of 124 mg (0.29 mmol) of BOC-(inip)-DβNal (from Example 29,step A), 84.5 mg (0.54 mmol) of 2-aminomethylnaphthylene hydrochloride(step A), 67 mg (0.348 mmol) of EDC, 47 mg (0.348 mmol) of HOBt, and 140μL of NMM in 5 ml of DMF was stirred overnight at ambient temperature.The reaction mixture was partitioned between ethyl acetate and water andthe separated organic phase washed successively with 1 N sodium hydrogensulfate, 1 N sodium bicarbonate, brine, dried over magnesium sulfate,filtered, and evaporated. The crude product was dissolved in 4 ml ofDCM/TFA (1:1), stirred for 2 h, and reconcentrated to give 160 mg ofcrude product. An 85 mg aliquot was purified by HPLC (1×50 cm, VydacC-18, 23 to 38% acetonitrile in water over 60 min, 0.1% TFA, 9 mL/min,rt=45 min) to give 4.6 mg of the title compound. MS (electrospray, M+H)466.0.

EXAMPLE 35 ##STR84## (inip)-DβNal-(D-Tryptophanol), TFA salt

Step A: BOC-DβNal-DTrp-(O-resin)

A 1.5 g sample of BOC-DTrp-(O-resin) (0.5 mmol/g, 0.75 mmol) wasdeblocked (TFA/DCM (1:1) containing 1 g/L of indole as a scavenger),washed, neutralized, and coupled with 0.71 g (3 eq) of BOC-DβNal, 1.02 g(3 eq) of BOP, 0.30 g (3 eq) of HOBt, and 0.37 mL (4.5 eq) of NMM for1.5 h, according to the general BOC procedure, givingBOC-DβNal-DTrp-(O-resin) (ninhydrin negative).

Step B: BOC-(inip)-DβNal-DTrp-(O-resin)

The above sample of BOC-DβNal-DTrp-(O-resin) was deblocked, washed, andcoupled with 0.52 g (3 eq) of N-BOC-isonipecotic acid, 1.02 g (3 eq) ofBOP, 0.30 g (3 eq) of HOBt, and 0.37 mL (4.5 eq) of NMM for 1 h, giving1.76 g of the title compound after washing with methanol and drying invacuo (ninhydrin negative).

Step C: (inip)-DβNal-(D-Tryptophanol), TFA salt

The dry resin from step B (BOC-(inip)-DβNal-DTrp-(O-resin), 1.76 g) wassuspended in 50 mL of THF under nitrogen and 4.80 mL of a 2.0 M solutionof lithium borohydride in THF added. After 1.5 h, 4.5 mL of HOAc wasadded dropwise over 10 min. After 30 min, the suspension was filtered,and the resin washed with MeOH. The combined filtrates were concentratedand partitioned between ethyl acetate and water (a few drops of HOAcadded). The organic phase was concentrated, and treated with 20 mL ofTFA/DCM (1:1) for 30 min. The TFA was removed in vacuo and the productreconcentrated from DCM (3×) to give 700 mg of an oil. A 100 mg aliquotwas purified by reverse phase HPLC (15-20μ, 300 Å, Vydac C-1, 1×50 cm,gradient: 23-38% acetonitrile (0.1% TFA) in water (0.1% TFA) in 60 minat 9 mL/min, rt=20 min) to give 23 mg of the title compound. MS(electrospray, M+H) 499.5.

EXAMPLE 36 ##STR85## (inip)-DβNal-[N-tryptaminyl]amide, TFA salt

Step A: BOC-(inip)-DβNal-[N-tryptaminyl]amide

A mixture of 100 mg (0.23 mmol, 1.0 eq) of N-BOC-(inip)-DβNal (fromExample 29, step A), 132 mg (0.69 mmol, 3.0 eq) of EDC, and 47 mg (0.35mmol, 1.5 eq) of HOBt in 15 ml of DCM/DMF (2:1) was stirred for 10 min,then 40 mg (0.25 mmol, 1.1 eq) of tryptamine and 40 μl (0.35 mmol, 1.5eq) of NMM were added. After 14 h at ambient temperature, the reactionmixture was partitioned between ethyl acetate and dilute citric acid andthe separated organic phase was washed successively with water,saturated sodium bicarbonate, brine, dried over sodium sulfate,filtered, and evaporated. The crude product (160 mg) was chromatographedon silica (ethyl acetate/hexane (70:30), Rf=0.5) to give 90 mg of thetitle compound as a crystalline solid. IR (cm⁻¹) 3342, 3289, 3057, 2977,2924, 2857, 1675, 136, 1556, 1436, 1224, 1164, 739. MS FAB, M+H) 569.3.

Step B: (inip)-DβNal-[N-tryptaminyl]amide, TFA salt

A solution of 70 mg of B-BOC-(inip)-DβNal-[N-tryptaminyl]amide (step A)in 4 ml of DCM was treated with 3 ml of TFA and stirred for 1 h. Theconcentrated crude product was purified by HPLC (1×50 cm, Vydac C-18, 20to 35% acetonitrile in water over 60 min, 9 mL/min, 0.1% TFA, rt=30-48min) to give 32 mg of the title compound. MS (electrospray, M+H) 469.4.

EXAMPLE 37 ##STR86## (inip)-DβNal-DTrp-Phe-Lys-amide, TFA salt

The title compound was prepared in an identical fashion to(inip)-DβNal-DβNal-Phe-Lys-amide, TFA salt (Example 1, method B) withthe only change being substitution of BOC-D-Tryptophan for BOC-DβNal inthe third coupling (step C). Washing, drying, and cleavage as per thegeneral BOC protocol above gave 250 mg of a powder. A 102 mg aliquot waspurified by reverse phase HPLC (15-20μ, 300 Å, Vydac C-18, 1×50 cm,gradient: 23-38% acetonitrile (0.1% TFA in water (0.1% TFA) in 60 min at9 mL/min, rt=32 min) to give 23 mg of the title compound. MS(electrospray, M+H) 812.4.

EXAMPLE 38 Anterior Pituitary Cell Assays ("Pit"Cell Assays)

Dispersion

Adult female Sprague-Dawley (160-180 g., Charles River) rats weregroup-caged in a 12:12 light:dark cycle with food and water available adlibitum. Pituitaries from ten rates were removed, the posteriorpituitary discarded and the anterior pituitary placed in Hanks' BalancedSalt Solution (HBSS: without calcium chloride, w/o magnesium chloride,w/o magnesium sulfate; Gibco) containing 20 mM HEPES (Gibco) and 100U/ml penicillin streptomycin (PS; JRH Biosciences). Under sterileconditions, pituitaries were rinsed twice then minced into smallfragments with a razon blade. Fragments were resuspended in 5 ml ofHBSS/HEPES containing 20 mg collagenase (Serva 17449) and 200 ml of 1mg/ml DNase (Sigma) for a 40 min. incubation in a 37° C. gyrotory waterbath shaker (New Brunswick Scientific Model G76; setting 10). After theincubation, fragments were triturated to yield small clumps and singlecells. The cells were centrifuged 1000×g. for 5 min, resuspended,counted and plated at a final cell concentration of 100,000 cells/ml.Incubation media was DME low glucose media w/NaHCO₃ (Gibco) containing20 mM hepes, 100 U/ml PS and 10% FBS (Hyclone A-111-L). Cells wereplated at 0.5 ml per well in 48 well plates (Falcon) and incubated at37° C. in 5% C02 for three days. For challenges to determine release ofother pituitary hormones cells were plated at 200,000 cells per ml with2 ml per well of a 6-well plate (Corning).

Challenge

A (inip)bbFK--NH₂ stock (or other GHRP) concentration of 1 mM was madein DMSO and diluted with warmed media approximately 30 min prior to use.The highest concentration of DMSO in media was 0.1%. Stock solutions (1mM) of rat GHRH, somatostatin (Sigma) and GHRP antagonist HwkWfK weremade fresh in media and diluted appropriately. The media used in allchallenges and washing steps was DME low glucose with 20 mM Hepes, 100U/ml PS, 10% FBS. Media was warmed to 37° C. and gassed by placing itinto the incubator prior to challenge. On day three, the media wasdiscarded and fresh media (approximately 1 ml) added for the first ofthree washes. After the last wash, the plate was placed back in theincubator for a 15 min pre-incubation. Then cells were washed 2× (withwarmed and gassed media) and fresh 0.5 ml media were added for thesecond 15 min. pre-incubation. After the second pre-incubation cellswere washed 2× as above and 0.5 ml of control and test solutions wereadded for a final 15 min incubation. After this incubation, the mediawere removed for subsequent GH ELISA.

GH ELISA

A two-site ELISA was used to determine rat GH concentration in themedia. Briefly, goat anti-rat GH antibody (lot# 19164-20) was used tocoat Nunc immunoplates overnight. After blocking and washing, standard(rat GH reference preparation: Parlow) and challenge media is diluted1:20 prior to GB assay were added for a 1 hr room temperatureincubation.

Statistics

The mean for each group was determined an analyzed by one-way analysisof variance with a post-hoc Student-Newman-Keuls. Significance isdefined as P<0.05. The EC₅₀ was calculated using a 4-parameter curve-fitprogram (Kaleidagraph). Three to four independent EC_(50s) were used toderive the means and SEM.

RIA of Pituitary Hormones

LH, FSH, TSH, and Prolactin were determined with commercially availablekits from Amersham, and ACTH levels were determined by a RIA kit fromICN.

Calcium Flux Experiment

Pituitary cells were plated on fibronectin (Collaborative Research)coated two-chambered slide wells (Nunc). After four days in monolayerculture cells were rinsed three times with HBSS (Gibco) in 1% BSA and 15mM HEPES and then incubated for 30 min at 37° C. with 5 μM Indo-1 AM(Molecular Probes, Eugene) in HBSS also containing 1% pluronic F127(Molecular Probes). The cells were rinsed once and fresh media added forRT incubation. Cells were challenged within 30 min with 10 nM(inip)bbFK--NH₂, vehicle or 2.5 uM ionomycin (Sigma). Ca⁺⁺ flux wasimaged with a Meridian ACAS 570 using stage scanning at 21 secondintervals. Ca⁺⁺ bound Indo-1 was measured at 405±22 nm and Ca⁺⁺ freeIndol-1 was measured at 530±15 nm. The ratio of bound vs free Indo-1 wascalculated and corrected with a standard curve created under identicalinstrument settings (Grynkiewicz G., M. Peonie, R.T. Tsien. A newgeneration of Ca⁺⁺ indicators with greatly improved flourescenceproperties. Journal of Biological Chemistry 260: 3440-3450, [1985]).

EXAMPLE 39 In vitro and In vivo Biological Data

Biological data for selected prior art compounds is provided in Table II

                  TABLE II                                                        ______________________________________                                                                                   Rat IV                               Literature   "Pit" Cell   ED.sub.50                                           code Structure C-term EC.sub.50 (nM) SE n (μg)                           ______________________________________                                               Y w w F     amide   1000        3                                        "GHRP 6" H w A W f K amide 6.2 1.5 5 1.0                                      "GHRP 2" a b A w f K amide 1.0 0.2 3  0.35                                     -- (Ava) b A W f K amide 0.2  0.03 3 1.5                                     "GHRP 1" A H b A W f K amide 1.4  2                                           L-692,429 benzo-fused  26.2  5.3 5 100                                         lactam                                                                       L-692,585 benzo-fused  10.6  4.0 4  10                                         lactam                                                                     ______________________________________                                    

The following selected in vitro and in vivo biological data forcompounds represented by formula IV is provided in Table III

                  TABLE III                                                       ______________________________________                                          #STR87##                                                                       -                      PIT Cell        RAT IV                                Structure C-term EC.sub.50 (nM) SE n ED.sub.50 (μg)                      ______________________________________                                        Y w w F K     amide   1000          3                                           H w w F K amide 25.8 3.9 3  50                                                H b w F K amide  6.8 1.2 3 4.2                                                H w b F K amide 15.0 5.0 3  50                                                H b b F K amide  2.4 1.2 3 15.4                                               G w w F K amide  2.1 0.3 3 2.5                                                (Ava) w w F K amide 13.0 2.6 4 5                                              a w w F K amide  4.1 2 3  1.47                                                G b w F K amide  0.8  2 4.8                                                   (Ava) b w F K amide  4.6 1.5 6 11.8                                           H w w nmF K amide 37.3 11.3 3                                                 G b b F K amide 14.9  2 100                                                   bA b b F K amide  4.8 3.3 3 6.7                                               (Ab) b b F K amide  0.4 0.1 3  1                                              (Ava) b b F K amide  2.8 0.4 4 2.6                                            (ahx) b b F K amide 1000  2 250                                               (ahp) b b F K amide 34.3 8.2 3 100                                            (nba) b b F K amide  2.8  2 100                                               (inip) b b F K amide  0.18 0.04 3 0.2                                         (pyc) b b F K amide >300  4 200                                               (pac) b b F K amide  100  2                                                   (Ava) b b F G amide  7.2 1.9 4  50                                            (Ava) b b F N amide  7.7 4.5 3 200                                            (Ava) b b F n amide  10  1 12.5                                               (Ava) b b F k amide  17  1 33.3                                               (Ava) b b F P amide  51  1  50                                                    b b F K amide 1000  2                                                     (Ava) b b F K Y amide 13.6 2.6 3  20                                          (Ava) b nmb F K amide  3.8 1.4 3 100                                          (Ava) nmb b F K amide  1.8 0.6 3 0.4                                          (Ava) b b B K amide  0.7  1  50                                               (Ava) b b hF K amide  5.0 1.7 3  50                                           (Ava) a b F K amide >300  2                                                   (Ava) b b chA K amide 44.3 9.5 3                                              (Ava) b b (Pg) K amide >100  1                                                (Ava) b b (pG) K amide >100  1                                                (inip) b b npA K amide  0.1  2  1                                             (Ava) b b A K amide >150  2                                                   (Ava) b a F K amide >300  3                                                   a b b F K amide  3.5 1.5 3  10                                                A b b F K amide  2.1 0.8 4  10                                                (mab) b b F K amide  1.4  1 0.7                                               (inip) nmb nmb F K amide  1.4  1                                              (inip) nmb b F K amide  0.27 0.06 4 0.4                                       (inip) b nmb F K amide  0.2  2  1                                             (inip) b b F K Y amide  0.43 0.01 4  1                                        (inip) b b F bA amide  0.2 0.05 4  2                                              nmb b F K amide >100  1 1000                                              (Ab) nmb b F K amide  0.5  2  1                                               (inip) nmb w F K amide  0.2  2 0.2                                            (inip) b b F K GGSGGSY amide  0.4  2                                          (inip) nmb b F dam   6.0  1                                                   (inip) nmb b F mor   0.8  1  10                                                   nmb b Y K amide 1000  4 500                                               (Ava) b b npA K amide 1000  1                                                 (cho) b b F K amide 1000  1                                                   (Ab) b b B ram   2.4 1.1 3  10                                                (inip) b b f dam   12  1                                                      (inip) b b F dam   12  1  10                                                  (inip) b b F mam   7.0  1                                                     (inip) b b F mam   2.0  1  2                                                  (inip) b b F tbm   10  1  10                                                  (inip) b b Y(I) K amide                                                       (inip) b b Y K amide  0.2  1                                                  (inip) b b F K Y(I) amide  0.69 0.54 3                                        (inip) b w F K amide  1.1 0.5 3  0.070                                      ______________________________________                                    

The following selected in vitro and in vivo biological data forcompounds represented by formula III is provided in Table IV

                  TABLE IV                                                        ______________________________________                                          #STR88##                                                                       -                    PIT Cell          RAT IV                                Structure C-term ED50 (nM) SE n ED50 (μg)                                ______________________________________                                        H w w F    amide    1000           4                                            (Ava) f f F amide 1000  1                                                     (Ava) f w F amide >300  3 1000                                                (Ava) b b F amide 17.1  5.7 3 100                                             (Ava) b b fem  >200  4                                                        (inip) b b bam  18.5   2 3.3                                                  (Ava) b f K amide >300  4                                                     (inip) b b fem   2.04 0.9 3                                                   (inip) b b F amide  0.28 0.07 5 1.7                                           (inip) nmb b F amide 0.5 0.2 4  5                                             (inip) nl b F amide  101  1                                                   (inip) b nl F amide  72  1                                                    (inip) b b nL amide 4.3  2 3.3                                                (inip) b b P amide   9  1  5                                                  (inip) b b amF amide 0.5  1  5                                                (inip) b b nmF amide 0.6  2  5                                                (inip) nmb b F acid 1.4  1  5                                                 (inip) nmb b F Me ester 1.4  2 10                                             (inip) nmb B F  >100  1                                                       (inip) b b feg amide  0.25 0.19 3  1                                          (inip) b b feb amide 0.3  2  0.67                                             (inip) b b fbd  0.9  2  0.67                                                  (inip) b b hcF amide 0.2 0.09 3 2.5                                           (inip) hf b F amide  27  1                                                    (inip) b hf F amide  14  1 10                                                 (inip) hf hf F amide >1000   1                                                (inip) b f F amide  79  1                                                     (inip) b b (Tic) amide  12  1 10                                              (inip) b (tic) F amide >1000   1                                              (Ava) nmb b F amide 1.6  1 20                                                 (Ab) nmb b F amide 0.2  2 20                                                  (inip) b b f amide 0.6  1 20                                                  (inip) nmb B F acid   7  1 100                                                (inip) b b npA amide 0.2  1  5                                                (inip) b b B amide 0.1  2 50                                                  (Ab) b b F amide 3.1  1 10                                                    (inip) b b Pol  0.2 0.02 3  5                                                 (inip) b chf F amide 2.7  1  5                                                (inip) chf b F amide 2.2  1 10                                                (inip) b nmb F amide   1  2  5                                                (inip) b b bA amide  10  1 10                                                 (inip) b b F Me ester 2.8  1  2                                               (inip) b b F acid 0.2  2  2                                                   (inip) w w F amide 0.9  2  5                                                  (inip) b w F amide 0.1  2  0.67                                               (inip) b b Abx amide  13  1 100                                               (inip) b Abx F amide >100  1                                                  (inip) b b tam   11  1 20                                                     (inip) b b ram   16  1  5                                                     (inip) b b pam   43  1                                                        (inip) b b cxa   14  1 10                                                     (inip) b b ppz   10  1 10                                                     (inip) b b ab amide  10  1 10                                                 (inip) b b apc   39  1                                                        (inip) b b api  2.7  2 25                                                     (inip) b b O amide 5.9  2  1                                                  (inip) b b o amide  10  1                                                     (inip) b b Y acid   5  1 10                                                   (inip) b nmb bam  0.3  2  1.67                                                (inip) nmb B bam  1000  1                                                     (inip) nmb b bam  n/d                                                         (inip) b nmb api  0.5  2  2                                                   (inip) b b Y(I) acid 0.1  2                                                 ______________________________________                                    

The following selected in vitro and in vivo biological data forcompounds represented by formula II is provided in Table V

                  TABLE V                                                         ______________________________________                                          #STR89##                                                                       -                    PIT Cell         RAT IV                                 Structure C-term EC.sub.50 (nM) SE n ED.sub.50 (μg)                      ______________________________________                                        (inip) b b amide    17.6      5.6 3                                             (inip) b Bmn  36  1 20                                                        (inip) b b acid 82  1                                                         (inip) b bol   2  2 10                                                        (inip) b b Me ester  5  1 100                                                 (inip) b mbm   7  1 20                                                        (inip) b Bol  100   4                                                         (inip) B Bol  1000   1                                                        (inip) B bol  1000   1                                                        (inip) b wol  10.6 6.2 3 0.8                                                  (inip) w wol   4  1 20                                                        (inip) w bol  1000   1                                                        (inip) b npe  11  2 500                                                       (inip) b men  12  2 500                                                       (inip) b man   9  1 500                                                       (inip) b tam  1000   1                                                        (Ava) b bol  190   1                                                          (inip) b nbol   3  1 20                                                       (amb) b bol  1000   1                                                         (inip) b miz  12  1                                                           (inip) b mbm Ac  90  2                                                        Boc (inip) b mbm  13  1                                                       (inip) b tra  100   2 100                                                   ______________________________________                                    

The following selected in vitro and in vivo biological data for the"retroinverso" compounds represented by formula V is provided in TableVI

                  TABLE VI                                                        ______________________________________                                          #STR90##                                                                       -                    PIT Cell         RAT IV                                 Structure C-term EC.sub.50 (nM) SE n ED.sub.50 (μg)                      ______________________________________                                        (dhc) B B bam       >200          3                                             (Ava) f B B K amide  36  1                                                    (Ava) f B B bam   22  1 100                                                   (Ab) b B B ram    2  2                                                        b B B ram  15.2   1 100                                                       Ac b B B ram  4.8  2 100                                                      (inip) B B api   96  2                                                        (inip) B B bam  1000  1                                                     ______________________________________                                    

EXAMPLE 40 GHRP Induced Growth Hormone Secretion: IntravenousAdministration

Immature weanling female Sprague Dawley rats were purchased from CharlesRiver Labs (Portage, Oreg.) and group housed with water and foodavailable ad libitum. When the rats were 24-30 days (weighing 50-90 g)they were anesthetized with pentobarbitone (4 mg in 0.5 ml,approximately 60 mg/kg) given by intraperitoneal injection. The ratswere then placed briefly on a heated pad, to distend their tail veins,and given an intravenous tail vein injection of the peptides 20 minutesafter receiving the anesthetic. The intravenous injection was of 0.1 mlusing a 1 ml syringe. The injections contained graded doses of peptidesor the vehicle (vehicle for all peptides given intravenously was abuffer of 20 mM sodium acetate, 45 g/l mannitol, pH 5.0). Ten minutesafter the intravenous injection blood was taken by cardiac puncture,using a 3 ml syringe, and the rats were then sacrificed.

The blood was then clotted on ice, centrifuged, serum decanted andfrozen for subsequent analysis using the rat GH ELISA describedelsewhere in the application. For the rat GB ELISA the serum was diluted1:50 or 1:250, depending on the expected serum GH concentrationachieved, and assay in duplicate.

EXAMPLE 41 GHRP Induced Dose Dependent Weight Gain in Rats

Methods

Forty normal Sprague Dawley female rats (Supplier, Charles River, 90days of age, average weight 200 g) were group housed in a roomcontrolled for temperature and lighting and fed a standard pelletted ratdiet and tap water ad libitum. The rats were weighed on the day ofsurgery (see below) and randomized into 5 groups of 8/group using agrouping program.

The GHRP (inip) b b F K--NH₂, was dissolved in sodium acetate (20 mM)buffer (pH 5.0) containing mannitol (45 g/l) at 8 g/l, 1.6 g/l and 0.33g/l. Rat GHRH (1-43) was dissolved in the same buffer at 25 g/l. Osmoticminipumps (Alza, Palo Alto, model 2002, pump rate 0.52 μl/hr for 14days, fill volume 230 μl) were filled with these solutions (1/rat for(inip) b b F K--NH₂ and 2/rat for rat GHRH); a fifth set of pumps werefilled with the sodium acetate buffer. All the pumps were primed bybeing incubated in isotonic saline overnight in a refrigerator.

The next day these osmotic pumps were inserted into rats. To do this therats were anesthetized with ketamine/xylazine (62.5 and 12.5 mg/kg/rat,respectively, by i.p. injection). The dorsal neck was then shaved,swabbed with betadine solution and cleaned with alcohol. An incision wasthen made on the dorsal neck and the a subcutaneous pocket createdcarefully by blunt dissection. The pumps were then inserted into thepocket with the end of the pump delivering the solution positioned awayfrom the incision. The incision was then closed with wound clips, therat placed on a heated pad and when ambulatory was returned to its homecage.

The rats were then weighed every day and on day 14 they were sacrificedusing inhalation of carbon dioxide. They were then bled from the heartand organs taken. The pituitary, spleen, heart, kidney, liver, andthymus were taken and weighed while the tibias were removed and placedin 10% formalin for subsequent histological evaluation. To do this thetibias were sectioned longitudinally and the width of the epiphysealplate was measured using a microscope fitted with an ocular micrometer.

Serum chemistries were measured by standard automated procedures. Seruminsulin-like growth factor-1 (IGF-1) was measured by radioimmunoassay,using an antibody raised in rabbits, after acid ethanol extraction toremove the IGF-1 binding protein.

Statistical significance was gauged by analysis of variance, which ifsignificant (p<0.05) was followed by a Duncan's New Multiple Range Testto test for differences between the individual treatment groups. Dataare presented as mean±standard error of the mean with 8 rats per group.

The body weight gains plotted against time for the 5 treatment groupsare shown in FIG. 19. Both (inip) b b F K--NH₂ and rat GHRH inducedsignificant body weight and organ weight gain compared to the vehicletreated rats.

EXAMPLE 42 Comparison of SC Injections and SC Infusions

Methods

Forty normal Sprague Dawley female rates (supplier Charles River, 150days of age, average weight 280 g) were group housed in a roomcontrolled for temperature and lighting and fed a standard pelletted ratdiet and tap water ad libitum. The rats were weighed on the day ofsurgery (see below) and randomized into 5 groups of 8/group using agrouping program.

GHRP (inip) b b F K--NH2 was dissolved in a sodium acetate (20 mM)buffer (pH 5.0) containing mannitol (45 g/l) at 8 g/l and 1.6 g/l tofill the minipumps and at 0.5 and 0.1 g/l for the injection solutions.Osmotic minipumps (Alza, Palo Alto, model 2002, pump rate 0.52 μl/hr for14 days, fill volume 230 μl, once per rat) were filled with thesesolutions; a fifth set of pumps were filled with the sodium acetatebuffer. All the pumps were primed by being incubated in isotonic salineovernight in a refrigerator.

The next day osmotic pumps were inserted into all rats. To do this therats were anesthetized with ketamine/xylazine (62.5 and 12.5 mg/kg/rat,respectively, by i.p. injection). The dorsal neck was then shaved,swabbed with betadine solution and cleaned with alcohol. An incision wasthen made on the dorsal neck and the a subcutaneous pocket createdcaudally by blunt dissection. The pumps were then inserted into thepocket with the end of the pump delivering the solution positioned awayfrom the incision. The incision was then closed with wound clips, therat placed on a heated pad and when ambulatory was returned to its homecage.

The treatment groups were;

1) Excipient pump, excipient injections 2 times a day.

2) b b F K--NH₂ pump (100 μg/day), excipient injections 2 times a day.

3) (inip) b b F K--NH₂ pump (20 μg/day, excipient injections 2 times aday.

4) (inip) b b F K--NH₂ injections 50 μg 2 times a day.

5) (inip) b b F K--NH₂ injections 10 μg 2 times a day.

The rats were then weighed every day and injected twice daily witheither excipient or the two doses of (inip) b b F K--NH₂. On day 14 theywere sacrificed using inhalation of carbon dioxide. They were then bledfrom the heart and organs taken. The rats were skinned and evisceratedto weigh the amount of skin, muscle and bone (the carcass). Thepituitary, spleen, heart, kidney, liver, thymus and the soleus musclewere also taken and weighed while the tibias were removed and placed in10% formalin for subsequent histological evaluation. The Tibias weresectioned longitudinally and the width of the epiphyseal plate wasmeasured using a microscope fitted with an ocular micrometer.

Serum chemistries were measured using standard automated techniques.Serum insulin-like growth factor-1 (IGF-1) was measured byradioimmunoassay, using an antibody raised in rabbits, after acidethanol extraction to remove the IGF-1 binding protein.

Statistical significance was gauged by analysis of variance, which ifsignificant (p<0.05) was followed by a Duncan's New Multiple Range Testto test for differences between the individual treatment groups. Dataare presented as mean±standard error of the mean with 8 rats per group.

(inip) b b F K--NH₂ at 20 and 100 μg/day, delivered by both injectionand infusion, induced significant body weight gain compared to vehicletreated rats. The dose-related nature of the body weight gains toinjections of (inip) b b F K--NH₂ can be seen in FIG. 20. In contrastthere were similar weight gains in response to infusions of both 20 and100 μg/day of (inip) b b F K--NH₂. In addition there were very differentpatterns of weight gain in response to infusions or injections of 100μg/day of (inip) b b F K--NH₂ as can be seen in FIG. 21.

EXAMPLE 43 Combination GHRP and IGF-1 Treatment of Obese Rats

Methods

Forty-eight (48)obese male Zucker Diabetic Fatty (ZDF) rats (GeneticModels Inc., Indianapolis, Ind. 46268) 6 weeks of age were group housedin a room controlled for temperature and lighting and fed a standardpelletted rat diet and tap water ad libitum. The rats were weighed onthe day of surgery (see below) and randomized into 6 groups of 8/groupusing a grouping program. Ten lean ZDF rats served as an additionalcontrol group.

GHRP (inip) b b F K--NH₂ was dissolved in a sodium acetate (20 mM)buffer (pH 5.0) containing mannitol (45 g/l) at 0.5 g/l. This GHRP wasgiven by sc. injection twice daily, each dose of 100 μl thereforecontaining 50 μg/injection or 100 μg/day.

Recombinant human IGF-1 (rhIGF-1) at 13.8 mg/ml in acetate buffer wasloaded into osmotic minipumps (Alza, Palo Alto, model 2ML4, pump rate2.29 μl/hr for 28 days, fill volume 2064 μl). Other pumps were filledwith acetate buffer. The pumps were primed by being incubated inisotonic saline overnight in a refrigerator. The delivered dose ofrhIGF-1 was therefore 758 μg/day. Recombinant human growth hormone(rhGH, Lot R9092AX, Genentech Inc.) was diluted in sterile water to 2.5g/l and a 100 μl injection given twice daily (250 μg/injection, or 500μg/day).

The next day the osmotic pumps were inserted into rats. To do this therats were anesthetized with ketamine/xylazine (62.5 and 12.5 mg/kg/rat,respectively, by i.p. injection). The dorsal neck was then shaved,swabbed with betadine solution and cleaned with alcohol. An incision wasthen made on the dorsal neck an the a subcutaneous pocket createdcaudally by blunt dissection. The pumps were then inserted into thepocket with the end of the pump delivering the solution positioned awayfrom the incision. All rats not receiving rhIGF-1 containing pumps wereimplanted with pumps delivering the acetate buffer excipient. Theincision was then closed with would clips, the rat placed on a heatedpad and when ambulatory was returned to its home cage.

The rats were then weighed every day, and injected twice daily witheither active drug (GH or GHRP) or vehicle excipient. On day 24 bloodwas withdrawn after a 4 hour fast and 1.5 U/kg of regular insulin wasinjected i.p. and a second blood sample taken 30 minutes later. The ratswere then sacrificed using carbon dioxide, bled from the heart, andorgans taken. Serum glucose was measured by standard automatedprocedures.

Statistical significance was gauged by analysis of variance, which ifsignificant (p<0.05) was followed by a Duncan's New Multiple Range Testto test for differences between the individual treatment groups. Dataare presented as mean±standard error (SE) of the mean with 8 rats pergroup.

Body Weight Gain

The body weight gains plotted against time for all treatment groups overthe whose study are shown in FIG. 22 and for the first 7 days for theGHRP (inip) b b F K--NH₂ and IFG-1 treatment groups are shown in FIG.23. The basal blood glucose values plotted against time for all thetreatment groups for the entire experiment are shown in FIG. 24 and theblood glucose responses to an intravenous insulin challenge at the endof the experiment are shown in FIG. 25.

EXAMPLE 44 Combination GHRP and IGF-1 Treatment of Normal Rats

Methods

Sixty normal adult female SD rats (Supplier, Charles River, 120 days ofage, 250 to 320 g) were group housed in a room controlled fortemperature and lighting and fed a standard pelletted rat diet and tapwater ad libitum. The rats were weighed on the day of surgery andrandomized into 12 groups of 5/group using a grouping program.

The GH secretagogues (GHRPs and GHRH) were dissolved in a sodium acetate(20 mM) buffer (pH 5.0) containing mannitol (45g/l) at 0.5 g/l. The GHsecretagogues were given by sc. injection twice daily, each dose of 100μl. Different doses of the molecules were given based on there potencyin the IV assay (for example L-692,585 was given at 3-fold higher dosesas it was less the least potent of the secretagogues). Recombinant humanIGF-1 (rhIGF-1) at 2.5 mg/ml in acetate buffer was loaded into osmoticminipumps (Alza, Palo Alto, model 2ML1, pump rate 10.16 μ/hr for 7 days,fill volume 2086 μl). Other pumps were filled with acetate buffer. Thepumps were primed by being incubated in isotonic saline overnight in arefrigerator. The delivered dose of rhIGF-1 was therefore 610 μg/day.

The next day the osmotic pumps were inserted into rats. To do this therats were anesthetized with ketamine/xylazine (62.5 and 12.5 mg/kg/rat,respectively, by i.p. injection). The dorsal neck was then shaved,swabbed with betadine solution and cleaned with alcohol. An incision wasthen made on the dorsal neck and the a subcutaneous pocket createdcaudally by blunt dissection. The pumps were then inserted into thepocket with the end of the pump delivering the solution positioned awayfrom the incision. All rats not receiving rhIGF-1 containing pumps wereimplanted with pumps delivering the acetate buffer excipient. Theincision was then closed with wound clips, the rat placed on a heatedpad and then when it was ambulatory returned to its home cage. Thetreatment groups were;

    ______________________________________                                         1) Excipient     2 injections/d                                                                           Excipient pump                                      2) Excipient 2 injections/d IGF-1 Pump                                        3) GHRH (300 μg/dose) 2 injections/d Excipient pump                        4) GHRH (300 μg/dose) 2 injections/d IGF-1 pump                            5) GHRP-6 (50 μg/dose) 2 injections/d Excipient pump                       6) GHRP-6 (50 μg/dose) 2 injections/d IGF-1 pump                           7) (inip) b b F-NH.sub.2 (50 μg/dose) 2 injections/d Excipient pump        8) (inip) b b F-NH.sub.2 (50 μg/dose) 2 injections/d IGF-1 pump                                        9) (inip) b nmb bam (50 μg/dose) 2                                       injections/d Excipient pump                        10) (inip) b nmb bam (50 μg/dose) 2 injections/d IGF-1 pump                11) L-692,585 (150 μg/dose) 2 injections/d Excipient pump                  12) L-692,585 (150 μg/dose) 2 injections/d IGF-1 pump                    ______________________________________                                    

The rats were then weighed every day, and injected twice daily witheither active drug (GHRH or GHRP) or vehicle excipient). The rats weresacrificed using carbon dioxide, bled from the heart and organs taken.Serum chemistries were measured by standard automated procedures.

Statistical significance was gauged by analysis of variance, which ifsignificant (p<0.05) was followed by a Duncan's New Multiple Range Testto test for differences between the individual treatment groups. Dataare presented as mean±standard error of the mean with 8 rats per group.

Body Weight Gain

The body weight pains plotted against time for the groups treated onlywith the GH secretagogues are shown in FIG. 26. The responses to thecombination of the GH secretagogues and IGF-1 tended to be greater thanto IGF-1 alone (FIG. 27).

All references described herein are expressly incorporated by reference.

What is claimed is:
 1. A compound represented by structural FormulaIIIa-IIIi ##STR91## where Ar¹ and Ar² are each independently selectedfrom indolyl, ##STR92## n and o are independently 1, 2 or 3; R^(B),R^(C) and R^(D) are selected from the grouphydrogen, C₁ -C₆ alkyloptionally substituted with a group selected fromNR² R³, and phenyl-C₁-C₃ -NR² R³, and halo(F, Cl, Br, I)C₁ -C₆ alkyl; R¹ is selectedfromhydrogen, C₁ -C₆ alkyl, C(═O)--C₁ -C₆ alkyl, C(═O)--NR² R³,C(═NR²)--NR² R³, C(═O)O--C₁ -C₆ alkyl, and halo (F, Cl, Br, I)C₁ -C₆alkyl; R² and R³ are selected fromhydrogen, C₁ -C₆ alkyl, piperidinyl,and halo(F, Cl, Br, I)C₁ -C₆ alkyl; R² and R³ together with the N towhich they are bonded may form a 5- or 6-member heterocycle, optionallycontaining one additional hetero atom selected from O, S, and N whereany N is optionally substituted with R¹, any carbon is optionallysubstituted with R⁷ and where the heterocycle is optionally fused to aphenyl ring, optionally substituted with R⁴ ; R⁴ and R⁵ areindependently selected from the grouphydrogen halo (F, Cl, Br, and I),cyano, amino, amido, nitro, hydroxy, C₁ -C₄ perfluoroalkyl, and C₁ -C₃perfluoroalkoxy; R⁷ is selected from the groupCOOR², CONR² R³, cyano,NR² R³, NR² COR³, azido, nitro, hydroxy, C₆ -C₁₀ aryl optionallysubstituted withhalo (F, Cl, Br, and I), cyano, amino, amido, nitro,hydroxy, C₁ -C₄ perfluoroalkyl, and C₁ -C₃ perfluoroalkoxy; Y isselected from the groupC₁ -C₆ alkyl substituted with 1-2 R⁷, C₂ -C₆alkynyl optionally substituted with 1-2R⁷, C₂ -C₆ alkenyl optionallysubstituted with 1-2R⁷, C₁ -C₆ alkyloxy optionally substituted with1-2R⁷, and piperidinyl; ora pharmaceutically acceptable salt thereof. 2.The compound of claim 1 represented by structural Formula (IIIa)##STR93## where Ar¹ and Ar² are each independently selected fromindolyl, and ##STR94## R^(B), R^(C) and R^(D) are selected from thegroup hydrogen,C₁ -C₆ alkyl, C₆ -C₁₀ aryl--C₁ -C₆ alkyl, and halo (F,Cl, Br, I)C₁ -C₆ alkyl; R² and R³ are selected fromhydrogen, C₁ -C₆alkyl, piperidinyl, and halo (F, Cl, Br, I)C₁ -C₆ alkyl; R² and R³together with the nitrogen to which they are attached may form onoptionally mono- or di-substituted ring selected frompiperidinyl,pyrroylidinyl, pyrryl, imidazolyl, piperazinyl, and morpholinylwhere thesubstituents are selected from C₁ -C₃ alkyl; R⁷ is selected from thegroupCOOR², CONR² R³, cyano, NR² R³, NR² COR³, azido, nitro, hydroxy,and C₆ -C₁₀ aryl optionally substituted withhalo (F, Cl, Br, and I),cyano, amino, amido, nitro, hydroxy, C₁ -C₄ perfluoroalkyl, and C₁ -C₃perfluoroalkoxy; Y is selected from the groupC₁ -C₆ alkyl substitutedwith 1-2 R⁷, C₂ -C₆ alkynyl optionally substituted with 1-2R⁷, C₂ -C₆alkenyl optionally substituted with 1-2 R⁷, C₁ -C₆ alkyloxy optionallysubstituted with 1-2 R⁷, and piperidinyl; Y and R^(D) together with theN to which they are bonded may form a 5- or 6-member heterocycle,optionally containing one additional hetero atom selected from O, S, andN where any N is optionally substituted with R¹, any carbon isoptionally substituted with R⁷ and where the heterocycle is optionallyfused to a phenyl ring; ora pharmaceutically acceptable salt thereof. 3.The compound of claim 2 selected from the group consisting of ##STR95##a pharmaceutically acceptable salt thereof.
 4. A pharmaceuticalcomposition comprising a pharmaceutically acceptable excipient and thecompound of claim
 1. 5. A method for increasing the level of endogenousgrowth hormone in a mammal comprising administering to the mammal apharmaceutically effective amount of the composition of claim 4 to themammal.
 6. The method of claim 5, further comprising administering thecomposition in combination with a growth factor selected from the groupconsisting of growth hormone (GH), growth hormone releasing hormone(GHRH), insulin-like growth factor-1 (IFG-1) and insulin-like growthfactor-2 (IGF-2).
 7. A method for treating type II diabetes in a mammalin need of such treatment comprising administering to the mammal apharmaceutically effective amount of the composition of claim 4 to themammal.